Seeding nets with neutral spores of the red alga Porphyra umbilicalis (L.) Kutzing for use in integrated multi-trophic aquaculture (IMTA)

Nets in traditional Porphyra mariculture are seeded with conchospores derived from the conchocelis phase, and spend a nursery period in culture tanks or calm coastal waters until they reach several centimeters in length. Some species of Porphyra can regenerate the foliose phase directly through asexual reproduction, which suggests that the time, infrastructure, and costs associated with conchocelis culture might be avoided by seeding nets with asexual spores. Here, we present work from a short-term mariculture study using nets seeded with asexual spores (neutral spores) of a native Maine species of Porphyra. Porphyra umbilicalis (L.) Kutzing was selected for this proof of concept research because of its reproductive biology, abundance across seasons in Maine, and evidence of its promise as a mariculture crop. We studied the maturation, release, and germination of the neutral spores to develop an appropriate seeding protocol for nets, followed by development of a nursery raceway to provide an easily manipulated environment for the seeded nets. Neutral spores were produced throughout the year on the central Maine coast,however, there was a temporal variability in the number and survival of released neutral spores, depending upon thallus position in the intertidal zone. Small thalli were strictly vegetative, but most thalli reproduced by neutral spores- sexual reproduction was absent. Neutral spores germinated quickly at 10 and 15 'C, but germination was delayed at 5 degrees C. Unlike some algal zygotes and spores, neutral spores of R umbilicalis required light to germinate; however, irradiances of 25 and 100 mu mol photons M-2 S-1 were equally sufficient for germination. Rafts of seeded nets were deployed in Cobscook Bay, Maine, at two distances from salmon aquaculture pens and at a control site on a nearby, fallow aquaculture site (no salmon). There was no difference in nitrogen content of harvested thalli; however, both the density and the surface area of harvested thalli were different among the sites. The possible causes of these differences are discussed in the context of potential use of P umbilicalis in IMTA. (C) 2007 Elsevier B.V. All rights reserved.

海南岛蕨类植物的分类、区系地理与保育

摘要 "基于形态－地理学方法，通过野外调查，结合大量标本研究，在前人研究的基础上，对海南蕨类植物的分类进行了进一步修订;主要根据蕨类植物的现代地理分布，结合古生物学等有关资料，初步探讨了海南蕨类植物的区系性质与起源;根据IUCN2001年红色名录的等级和标准，对海南濒危蕨类植物的现状进行了初步评估，讨论了海南蕨类植物的受威胁原因，提出了有关保护的对策。主要结果如下： 1. 海南现有蕨类植物56科140属439个种及种下分类群（包括421种、15变种、2亚种和1变型），其中包括1个中国分布新记录和27个海南分布新记录，1新种――海南符藤蕨Teratophyllum hainanense;另有11个名称首次被处理为异名;澄清了海南假瘤蕨Phymatopteris hainanensis和圆顶假瘤蕨P. obtusa的模式问题，为滇桂三相蕨Ataxipteris dianguiensis、海南假瘤蕨P. hainanensis和浅杯鳞盖蕨Microlepia ampla指定了后选模式。 2. 海南蕨类区系具有以下特点：i. 以水龙骨科Polypodiaceae、金星蕨科Thelypteridaceae、铁角蕨科Aspleniaceae、叉蕨科Tectariaceae和观音座莲科Angiopteridaceae为表征科;ii. 明显的热带性质，科的97.5％、属的92.5％、种的83.6％为热带分布成分;iii. 很高的物种多样性与物种密度，但属内种系贫乏;iv. 与中南半岛的联系最为紧密，海南140个蕨类属中有136个与中南半岛共有，两地属的相似性系数达到87.2%;v. 海南蕨类区系就地起源于华夏古陆，起源时间可以追溯至早石炭世以前。 3. 海南439种蕨类（包括421种、15变种、2亚种和1变型）中，183种为常见蕨类，113种属于资料缺乏的种类（DD），47种属于近危（NT），53种属于易危（VU），37种属于濒危（EN），6种属于极危（CR）。海南的受威胁蕨类植物有96种，海南的绝大部分受威胁蕨类植物都生于保护区以内或得到有效保护的林区之内，已初步得到保护。导致海南96种蕨类受威胁的因素，除了植物本身的生物生态学特性和地理分布上的限制外，主要是人类活动的影响，特别是海南森林在上个世纪被大规模砍伐。为了保护这些受威胁植物，应加强保护区和林区的管理，实施就地保护，积极开展迁地保护和人工繁殖。 "

黑龙江嘉荫地区古新世孢粉植物群及古植被、古气候研究

植物是环境变化的重要指示物之一。晚白垩至早第三纪时期，全球生态系统发生了剧烈变化，研究这一时期植物对了解地质历史时期环境变化趋势, 认识和保护当今环境有重要意义。我国黑龙江省嘉荫县有这一时期的地层发育，其中乌云煤矿和白山头是主要的露头剖面。前人曾从地质时代、孢粉及植物化石角度对这两个剖面进行过研究，但是仍有部分问题尚存争议。在本项工作中，我们对两个剖面的孢粉样品进行了系统采样，部分孢粉类型同时在光镜和电镜下进行了拍照。我们还对存在争议的这两个剖面的地质时代进行了讨论。同时，首次用共存分析法对这两个剖面的在沉积时期的气候进行了整体及分段的定量重建。另外，我们结合中其它地点的气候重建工作对不同时期的纬度温度梯度进行了研究。 乌云煤矿和白山头两个乌云组剖面的古新世孢粉植物群的研究结果表明：乌云煤矿孢粉植物群主要是与榛属（Corylus），桤木属（Alnus），桦木属（Betula），榆属（Ulmus）及松属（Pinus）有亲缘关系的植物；白山头剖面的孢粉植物类型与乌云煤矿基本一致，但是其中针叶类植物的花粉所占比重较大。 根据乌云煤矿与白山头剖面的孢粉类型在地层中的分布，以及与其它古新世地层的对比，我们认为乌云煤矿与白山头含孢粉段的地质时代为古新世。 用共存分析法得到乌云组古新世气候参数有两组。孢粉类型的共存结果为：年均温14.8-14.9℃，年降水量816-1389mm；植物化石类型的共存结果为：年均温16.3-16.8℃, 年降水：1124-1623mm。对乌云煤矿与白山头两个剖面孢粉带中的孢粉类型的气候参数分别进行共存，结果表明，除乌云煤矿孢粉第三带到第四带外，乌云地区的年均温在整个沉积时期均呈上升趋势；年均降水量的变化趋势与年均温基本一致。 根据从古新世到现代不同地点气候定量重建的年均温参数，我们计算得到了不同时期纬度温度梯度变化的值：<0.1℃/古纬度（古新世）、 0.1℃/古纬度（始新世）、0.45℃/纬度（中新世）、0.55℃/纬度（上新世）。结合当今全球的平均温度梯度值（0.7℃/纬度），我们得出纬度温度梯度的值从古新世以来呈不断上升的趋势。这一结果显示65Ma 以来赤道与极地间的温度差异逐渐增大，同时也提示了全球温度可能呈下降趋势。

A combined phage display ScFv library against Myxobolus rotundus infecting crucian carp, Carassius auratus auratus (L.), in China

Immunological methods have been developed for the diagnosis of Myxobolus rotundus but their use has been limited for the prevention and therapy of this serious parasitic pathogen. Phage display antibody libraries are a powerful technique for the development of antibodies to molecules of interest and have advantages over traditional hybridroma approaches. In the present study, four antigen fractions related to M. rotundus were prepared and a combined phage display single-chain antibody fragments (ScFv) library was constructed against this parasite. Preliminary analysis indicated that a combined antibody library of about 2.08 X 10(5) individual clones and high diversity was generated. After four rounds of screening (bio-panning) against soluble spore protein prepared from lysed, intact, mature M rotundus spores, a strain monoclonal phage display ScFv, termed pCAN-6H9, with better affinity, was isolated. The pCAN-6H9 gene fragment was sequenced and analysed. The specificity of pCAN-6H9 was further demonstrated by dot-blot. In competition enzyme-linked immunosorbent assay, both the original and enriched phage-displayed ScFv repertoire showed significant inhibition of mouse anti-M rotundus serum binding to coated antigen, while the inhibition rate of monoclonal pCAN-6H9 phage particles was only 11.83%.

Humoral immune responses of the grouper Eninephelus akaara against the microsporidium Glugea epinephelusis

The humoral immune responses of grouper Epinephelus akaara to a natural infection with Glugea epinephelusis was studied by ELISA utilizing intact mature spores as the coated antigen. Results showed that a specific humoral immune response was elicited, but the intensity of infection (in terms of the number of cysts) was not related to the antibody level in naturally infected hosts. The differences in the antigenicity of intact mature spores and soluble spore proteins derived from cracked mature spores were also analyzed. Results suggested that similar antigen epitopes existed between the 2 groups. Additionally, antigen component patterns and the distribution of antigen with immunogenicity were investigated by using the western blot and the immunofluorescent antibody technique (IFAT). The new parasitic microsporidium has specific polypeptide patterns comparable to the reported fish microsporidians. The main antigenic substances are concentrated on the surface of spores, and are mostly located on the anterior and posterior end of the spore bodies. Most surface components of the G. epinephelusis spores are soluble, The potential role of the surface components in initiating infection was also discussed.

New data on Myxobolus longisporus (Myxozoa : Myxobolidae), a gill infecting parasite of carp, Cyprinus carpio haematopterus, from Chinese lakes

The original description of Myxobolus longisporus Nie et Li, 1992, the species infecting gills of Cyprinus carpio haematopterus L., is supplemented with new data on the spore morphology and pathogenicity. Spores are elongate pyriform with pointed anterior end, 15.7 (15.5-16.5) mum long, 6.7 (6-8) mum wide and 5.5 mum thick. Sutural ridge is straight and narrow. Mucus envelope is lacking. Two equal-sized elongate pyriform polar capsules are 8.5 mum long and 2.5 mum wide with convergent long axes. Polar filament coiled perpendicularly to the long axis of the capsule makes 9 (8-10) turns. Posterior end of polar capsules exceeds mid-spore by 15-20%. Cyst-like plasmodia are localised in the gill secondary lamellae. The infection is described in adult big host specimens. Gross lesions manifested as dark red colouration of gill tissues were restricted to the ventral part of the first gill arches. Remarkable site specificity (apical part of secondary lamellae) was observed in the course of development of microscopic lesions. M. longisporus is characterised also on the molecular level using sequences of SSU rRNA gene. Phylogenetic analysis based on these sequences has allowed clearer phylogenetic relationships to be established with other species of the genus Myxobolus sequenced to date.

Detection of Myxobolus rotundus (Myxozoa : Myxosporea) in skin mucus of crucian carp Carassius auratus auratus using a monoclonal antibody

Diagnosis of myxosporean Myxobolus rotundus infection was conducted by examining skin mucus from the infected crucian carp Carassius auratus auratus with a monoclonal antibody, MAb 2D12, raised previously against the parasite. A positive reaction was observed in skin mucus collected from infected fish, and spores and pre-spore stages of the parasite were identified by the MAb 2D12. It was also demonstrated that M. rotundus infection can be successfully detected by a simple method, enzyme-linked immunosorbent assay (ELISA), and that skin mucus collected from infected fish skin had a significantly higher optical density (OD) value than that from uninfected fish.

四川九顶山西坡及龙肘山维管植物区系和植物多样性研究

横断山地区是一个十分自然的植物区系地区，在中国植物区系分区中是作为泛北极植物区中国-喜马拉雅亚区中的一个地区，其种子植物区系具有丰富的科、属、种，地理成分复杂，特有现象和替代现象明显。该地区作为植物区系和生物多样性的研究热点地区，长期以来极受中外植物学家关注。横断山脉东缘是中国-喜马拉雅和中国-日本植物区系的交汇过渡区域，北部的岷江流域以及南部的金沙江流域，孕育了该区丰富的物种资源和植被资源。而岷江干热河谷和金沙江干热河谷的相似性和相关性，更为该区的植物区系和生物多样性南北的对比研究提供了有利的条件。 本研究选择的九顶山西坡和龙肘山分别位于横断山区北部和南部，九顶山属岷江流域而龙肘山属金沙江流域。本研究结合植物区系研究和生物多样性研究，对该区的植物资源进行调查。通过样带调查和样线踏查结合，大量详实的野外样方调查和标本采集，进行传统的区系研究和生物多样性研究。研究该区物种多样性的海拔梯度格局及其潜在的影响影子，并利用新的区系评估质量方法对九顶山西坡的植物区系质量进行定量的研究，以期能更为深刻的理解该区的植物资源，为该区的资源保护和利用提供合理可行的建议。主要研究结论如下： 1）九顶山西坡植物区系的性质和特点 经鉴定和统计，九顶山西坡共有1707 种维管植物，分属617 属和140 科,其中种子植物1616 种，分属572 属117 科。就科的分布区成分构成而言，该区系的热带成分与温带成分相当，热带成分略占优势，表明九顶山西坡的植物区系与热带植物区系有较强的联系。但是，在九顶山西坡属的分布区类型所占的比例上，温带成分远远超过了热带成分，本区的种子植物分布表现出明显的温带性质。且温带分布类型的许多物种组成了九顶山西坡植被的建群种和优势种，是本区系最重要的成分，充分体现了本区系的温带性质。 2）九顶山西坡不同植被带的生物多样性海拔梯度格局 基于对土门-断头崖、茶山-九顶山、雁门沟-光光山三条垂直植被样带的调查，我们发现九顶山西坡的生物多样性沿海拔梯度的变化呈现出一定的规律性，不同样带之间有一定差异。就三条样带的物种组成相似性来看，虽然土门－断头崖样带属于涪江水系，而茶山－九顶山样带和雁门沟－光光山样带属于岷江水系，但不同水系对该区物种组成的影响并不明显。三条样带中，草本层物种丰富度均远远大于灌木层和乔木层，而以乔木层物种丰富度最低；α－多样性指数随着海拔梯度的变化在土门-断头崖样带中呈现单一下降趋势，在茶山-九顶山样带表现为双峰模型，而在雁门沟-光光山样带则表现为不显著波动变化；均匀度指数在土门-断头崖样带呈现出单一下降的趋势，在雁门沟-光光山样带表现为凹形曲线，而在茶山-九顶山样带却无明显的变化规律。β－多样性指数在土门-断头崖样带和茶山-九顶山样带呈现出明显的波动状态，植被类型替代现象明显；而在雁门沟-光光山样带却并未有十分显著的转折点，因其水平植被带受到干扰，同海拔替代现象不显著。 3）九顶山西坡维管植物丰富度的海拔梯度格局 我们考察了九顶山西坡和两条垂直样带（土门－断头崖和雁门沟－光光山样带）的不同分类等级（包括科、属、种）和不同生活型物种（乔木、灌木、禾草、蕨类和其它草本）的丰富度沿着海拔梯度的分布。结果发现，物种的丰富度海拔梯度格局具有不同的模式，单一下降和中间膨胀格局依然是其主流。不同生活型的物种具有不同的丰富度格局，但是对于环境需求相似的类型具有较相似的丰富度格局。不同的丰富度格局可能由多因素导致，包括：气候，海拔跨度，面积，人为干扰等等。 4）九顶山西坡区系质量评估 我们尝试使用传统的区系质量评估方法对九顶山西坡的区系质量进行评估，并尝试使用一种新的区系质量评估体系对该区的区系进行评价。在九顶山西坡随着海拔梯度的上升，平均保守性系数在各条植被带中均呈现出逐渐上升的趋势。区系质量指数随着海拔的升高都表现为双峰模型，在植被交错区区系质量指数相对较高，而在海拔的两极，区系质量指数都很低。大部分地区使用新方法计算所得的加权平均保守性系数和区系质量指数都比传统方法计算的平均保收性系数和区系质量指数要高，说明在九顶山西坡的三条样带中，大部分地区都是那些保守性系数较高的物种占据优势，同时也表明九顶山西坡具有很高质量的区系和自然植被。 5）龙肘山种子植物区系的性质和特点 龙肘山种子植物区系的物种较为丰富，共有154 科，544 属，1156 种。科的优势十分明显，单种属和寡种属数量众多，说明本区系植物成分较为复杂、起源古老、物种多样性指数较高。地理成分复杂，分布类型多样，其中热带成分在总数量上高于温带成分，但是许多温带成分的属是该区植被的重要建群类群和优势类群，表现出明显的亚热带性质。 6）龙肘山生物多样性的现状和特点 在海拔梯度上，龙肘山地区无论是科、属、种的数量，还是不同等级分类单元之间的数量比，均呈现先升后降的趋势，并在中海拔地区达到峰值。物种多样性指数从总体上来说变化幅度不大，略有先升后降的趋势，在中海拔梯度物种多样性最高。乔、灌、草三层的多样性指数表现出乔木层<灌木层<草本层的特征；乔木层均匀度的变化很大，而灌木层和草本层均匀度的变化较小；灌木层均匀度的波动又强于草本层。β－多样性指数呈现单峰模式，中海拔地区最高。就龙肘山东、西坡物种多样性相比较而言，两者虽然在数值上交替上升，但是却体现出了较为一致的趋势，但西坡因受到干热河谷气候的影响，其平均气温要高于东坡，导致了东坡植物群落和物种的分布比西坡要低。在区系成分构成上，低山区的相同海拔段，西坡的热带亚热带成分所占的比例要比东坡高，这是因为西坡的平均气温比东坡稍高，导致了热带、亚热带物种分布更多。而随着海拔的上升，东、西两坡的气候、土壤等条件趋于一致，其植物区系成分的构成格局也趋于一致。 The Hengduan Mountain region is a very natural floristic region; it belongs toChina-Himalaya sub-region of Holarctic region in floristic subarea of China. The flora in this areais rich in family, genus and species; has a very complex composition of geographical elements;especially with high richness of endemic species and obvious substitution phenomenon. Thisregion as a hot-spot area of floristic and biodiversity, has fascinated biologists in the world for along time. The eastern range of Hengduan Mountain is the transition zone of China – Himalayaforest sub-region and China-Japan forest sub-region in floristic. The water systems are quitedifferent, Minjiang River in the north and Jishajiang River in the south grow quit different but alsoabundant plant species and vegetation resources. The similarity and correlativity of Minjiang River dry valleys and Jinshajiang River dry valleys have provided advantageous condition tocontrast flora and biodiversity between north and south. In the present study, the Jiuding Mountainlies in the north of Hengduan Mountain and belongs to Minjiang River, and the LongzhouMountain lies in the south of Hengduan Mountain and belongs to Jinshajiang River. In our study, we combined the methods of floristic research and biodiversity investigation toexplore the resources of plant species and vegetations; sampled with transects along the altitudinalgradients and also with transverse straps with similar elevation; collected the vascular plant specimen with sampling plots of ecology. We explored the plant species richness patterns alongaltitudinal gradients and discussed the underlying factors aroused these patterns; and used a novelmethod to assess the quality of Jiuding Mountain’s flora. All for a deeper comprehension of the plant recourses of this region; and provided feasible and reasonable suggestion for the protectionof resources. The results were as follows: 1 The characteristic of the flora of the west slope of Jiuding Mountain We had collected 1707 species of vascular plants belonging to 617 genera in 140 families inthe west slope of Jiuding Mountain，in which included 1616 seed plant species belonging to 572genera and 117 families. As for the composition of the areal types of the Families of seed plants,tropic components and temperate components are well-balanced, and percentage of tropicscomponents is higher than that of temperate ones for a litter bit. This shows the flora in the westslope of Jiuding Mountain has strong relationship with the tropic flora. But for the composition ofthe areal types of genera, temperate components have far exceeded the tropics ones, indicated thewhole flora with a conspicuous temperate character. Temperate components possess maximumproportion in the west slope of Jiuding Mountain, and many of them belong to constructivespecies and dominant species in the vegetation, are most important components in JiudingMountain’s Flora, also have embodied the temperate character of this area sufficiently. 2 Biodiversity patterns along altitudinal gradients in different vegetation transects in the westslope of Jiuding Mountain Based on the investigation of three vegetation transects (including Tumen-Duantouya transect,Chashan-Jiudingshan transect and Yanmengou-Guangguangshan Transect) in the west slope ofJiuding Mountain, we found the change of biodiversity along the altitude gradients displayedcertain regularity, but have differences among different transects. The three transects belong todifferent water systems; the Tumen-Duantouya transect belongs to Fujiang River, and the othertwo belong to Minjiang River. From the similarity of species compositions of different transects,we found different water system didn’t show obvious impact on the species composition. In all thethree transects, the species richness of herb layer was remarkably higher than shrub and tree layer,and the species richness of tree layer was the lowest one. With the increasing of the altitude, theline of α-diversity was monotonically decreasing curve in Tumen-Duantouya transect, andbimodal curve in Chashan-Jiudingshan transect, but in Yanmengou-Guangguangshan transectshowed a wave-like curve although not very obvious. Species evenness showed monotonicallydecreasing trends in Tumen-Duantouya transect, and very low at mid-altitude in Yanmengou-Guangguangshan transect, but in Chashan-Jiudingshan transect changed irregularly. Changes inβ-diversity corresponded with the transition of vegetation in the Tumen-Duantouya transect andChashan-Jiudingshan transect, and the curve of β-diversity along altitude had obvious turningpoint; but in Yanmengou-Guangguangshan transect had no obvious turning point, and thesubstitution phenomenon was not obvious, transverse vegetation straps distributed interlaced. 3 Richness patterns of vascular plant species along altitude in the west slope of Jiuding Mountain Direct gradient analysis and regression methods were used to describe the species richnesspatterns along the altitudinal for Mt. Jiuding, as well as separately for Tumen-Duantouya Transectand Yanmengou-Guangguangshan Transect. Altitudinal gradient of diversity of units at differenttaxonomic level (including Family, Genus and Species) and at different life form (including tree,shrub, pteridophyte, grass and other herb) were tested to find differences among the richnesspattern. We found altitudinal richness also shows different patterns, and both monotonicallydecreasing pattern and hump-shaped pattern can be founded in vascular species richness. Speciesin different life forms show different altitudinal patterns, but those species with similarrequirements to environmental conditions show similar richness patterns along altitudinalgradients. Different richness patterns can be aroused by different climate, different altitudinal span,area factor, anthropogenic factor and so on. 4 Floristic quality assessments in the west slope of Jiuding Mountain We used both the conventional method broadly adopted in the USA and the new one toassess the floristic quality in the west slope of Jiuding Mountain. The Mean Coefficient ofConservatism (MC) had the trend of increment along the altitudinal gradients. The FloristicQuality Index (FQI) was a bimodal curve with increasing of elevation; FQI got maximum valuesin the transition zones of different vegetations in the middle altitude, and had very low values atthe two end of elevation. In most areas of the west slope of the Jiuding Mountain, the resultscalculated using the new methods were higher than those using the conventional method. Thisindicated the dominant species of the communities had very high coefficients of conservatism inmost areas of Jiuding Mountain, and the communities are relatively kept pristine and the habitats very integrative. 5 The characteristic of the flora of Longzhou Mountain The flora of Longzhou Mountain has very abundant in species composition; there are about1156 species of seed plants belonging to 544 genera in 154 families. In which, twelve families with more than 20 species include totally 232 genera and 532 species, and form the majority of itsflora. The origin of its flora is old, monospecific genera and oligotypic genera amounts to 510 innumber, which constitute 93.75% of total number of genera. The geographical components arevarious in Longzhou Mountain, the majority of flora are temperate and pantropic ones. The tropiccomponents overtopped temperate components on genera quantity, but many temperatecomponents belong to constructive species and dominant species in the vegetation, and the wholeflora shows an obvious subtropical character. 6 Current situation and characteristic of biodiversity in Longzhou Mountain With the increasing of altitude, the number of species, genus, family and the ratios ofdifferent taxonomic levels all displayed a trend of descending after rising first, and peaked atmiddle height area. The change of α-diversity was not very acutely, with the trend of descendingafter rising first in some degree, the middle height area had highest α-diversity. As studying thetree layer, shrub layer and herb layer respectively, the Shannon-Wiener index was in followingorder: tree layer < shrub layer < herb layer; the change of evenness was more complicatedly thanthat of diversity, the tree layer changed acutely, but the shrub layer and herb layer fluctuatedsmoothly. Changes in β-diversity also showed the trend of descending after rising first. TheJaccard index and Cody index all peaked at the middle height forest area. As for the comparison ofplant diversity and evenness between the west and east slope, the numerical values ascendedalternatively, but the trend of changing was similar. The distribution of similar plant communitiesand species in east slope were lower than the west slope for the influence of Jinsha River DryValley. As for the composition of different floristic components, in lower altitude area of westslope, the tropic and sub-tropic plants had higher ratio than east slope’s and even could be equal tothe temperate plants. With the increasing of elevation, the floristic composition become morelikely between the east and west slope and temperate plants dominated the flora.

同源四倍体水稻三系亲本的遗传差异、细胞遗传学比较研究及低直链淀粉含量突变体Wx基因序列分析

同源四倍体水稻（2N=4X=48,AAAA）是由二倍体水稻（2N=2X=24,AA）通过秋水仙素诱导染色体加倍后得到的新品系，具有优良的抗病性以及较高的蛋白质含量。因此，在四倍体水平上挖掘水稻的增产潜力成为水稻育种的新手段。同源四倍体水稻具有很强的遗传可塑性和很弱的遗传保守性，利用其作为水稻远缘杂交的桥梁，从野生物种中不断地引进有益的基因，这将有助于杂交水稻的多代利用和固定水稻的杂种优势。但是迄今为止，还没有关于同源四倍体水稻遗传多样性，遗传背景的报道。目前世界关于同源四倍体水稻的研究主要集中在中国，主要研究方向为培育、筛选结实正常的亲本材料，配置和筛选结实率正常或接近正常的组合。经过几十年研究，虽然在材料构建，细胞学研究等方面取得了较大进展，但同样由于结实率低的瓶颈问题未解决，而使多倍体水稻育种未能取得实质性进展。而近年来一些关于同源四倍体水稻低结实率机理的细胞学研究也由于缺乏统计学数据而缺乏说明性。本文用SSR标记，对选取的36个结实率正常同源四倍体水稻三系亲本和14个来源二倍体亲本，分析他们的遗传差异和群体遗传结构。本文还利用我们培育的高、低结实率的同源四倍体水稻恢复系、优良保持系和杂种F1及二倍体对照为材料，进行系统深入的细胞遗传学研究，进一步探讨同源四倍体水稻有性传递后代的发育过程，探索分裂期染色体行为特征与遗传性状稳定性的关系，为进一步选育多倍体水稻品种并将其应用于生产提供理论依据。同源四倍体水稻突变株D4063-1直链淀粉含量比来源二倍体明恢63下降一半,即其直链淀粉含量为5.23%，为研究其直链淀粉含量下降的原因，本文还根据普通水稻Wx基因设计引物，扩增测序获得了D4063-1Wx基因的全序列，与已报道Wx基因进行比对分析，并根据D4063-1和籼稻、粳稻的序列差异并根据D4063-1在该片段上的特征序列位点设计了用于识别D4063-1的寡核苷酸片段,为快速、准确的鉴别低直链淀粉的D4063-1创造了条件。 SSR标记具有基因组分布广泛、数量丰富、多态性高、容易检测、共显性、结果稳定可靠、实验重现性好、操作简单、经济、易于高通量分析等许多优点，被认为是用于遗传多样性、品种鉴定、物种的系统发育、亲缘关系及起源等研究的非常有效的分子标记。本研究选取了中国科学院成都生物所培育的同源四倍体和二倍体水稻亲本，并用36个微卫星标记进行了遗传差异和种群遗传结构分析。在50个品系中，我们观察到较高水平的多态性，每基因等位基因数（Ae）分布于2至6之间（平均值3.028），多态性信息含量（PIC）分布于0.04至0.76之间（平均值0.366）；期望杂合度（He）分布于0.04至0.76之间（平均值0.370），Shannon指数（I）分布于0.098至1.613之间（平均值0.649）。同源四倍体品系的等位基因数，期望杂合性和Shannon指数都比二倍体品系高。在供试50个品系中，较多材料均发现Rare基因，根据SSR多态性指数我们构建了同源四倍体和二倍体水稻的核心指纹库。F-统计值表明遗传差异主要存在于同源四倍体品系中（Fst=0.066）。聚类分析结果表明50个品系可以分为4个组。I组包括所有的同源四倍体和二倍体籼稻保持系，以及一个同源四倍体籼稻雄性不育系及其来源二倍体。II组仅包括IR来源的品系。III组比II组和IV组更复杂，包括同源四倍体和二倍体籼稻恢复系品系。IV组包括同源四倍体和二倍体粳稻品系。此外,由于等位基因及配子的遗传差异,同源四倍体与二倍体品系中存在单位点和双位点的遗传差异。分析结果表明,二倍体和四倍体水稻基因库的不同,其中遗传变异可以区分四倍体与二倍体水稻。同源四倍体水稻具有长期而独立的遗传性，我们能够选育并得到与二倍体亲本相比有特殊优良农艺性状的品系。 本研究以高结实率的同源四倍体水稻恢复系DTP-4、D明恢63及优良保持系D46B为材料进行农艺性状及细胞遗传学比较研究。DTP-4、D明恢63及保持系D46B的的染色体组成均为2N=4X=48，花粉母细胞具有较为理想的减数分裂行为，配对染色体的比率在99%以上，这与理论染色体组构成相符。DTP-4和D明恢63PMC减数分裂各个时期单价体和三价体的比例都非常低，而在MI， PMC观察到较多的二价体和四价体且四价体多以环状形式出现，其最大频率的染色体构型分别为12II 6IV和10II 7IV。恢复系DTP-4和D明恢63在MI四价体频率分别为2.00/PMC和2.26/PMC，而保持系D46B在MI四价体频率为6.00/PMC，极显著地高于恢复系品系，表明保持系D46B具有更好的染色体配对性质；AI保持系D46B的染色体滞后频率为10.62%，远低于恢复系材料DTP－4和D明恢63的19.44％和23.14％，接近二倍体对照明恢63的7.30%水平；TI保持系D46B具有比恢复系更低频率的微核数。而在TII，D46B的正常四分小孢子比率不但高于恢复系品系甚至高于二倍体对照。对高低结实率的同源四倍体水稻恢复系和杂种F1代的花粉育性，结实率和细胞遗传学行为进行了比较研究。DTP-4, D明恢63, D46A´DTP-4和D46A´D明恢63的花粉育性和结实率比D什香和D46A´D什香显著提高。减数分裂分析的结果表明，DTP-4，D明恢63，D什香，D46A´DTP-4，D46A´D明恢63和D46A´D什香其减数分裂染色体构型分别为：0.05I +19.96 II (9.89棒状+10.07环状) +0.01III + 2.20 IV, 0.11I +19.17 II (8.90 棒状+10.37 环状) +0.09III + 2.26 IV + 0.01 VI, 1.33I +9.46 II (4.50 棒状+4.96 环状) +0.44III + 6.02 IV + 0.09VI + 0.09 VIII, 0.02I +14.36 II (6.44 棒状+7.91 环状) +0.01III + 4.80IV + 0.01VIII, 0.06 I +17.67 II (11.01 棒状+6.67 环状) +0.06 III + 3.10 IV + 0.01 VI and 1.11 I +11.31 II (5.80 棒状+5.51 环状) +0.41 III + 5.63 IV+0.03VI+0.03VIII。在同源四倍体水稻恢复系和杂种F1代材料中，最常见的染色体构型为16II +4IV和12II +6IV。在减数分裂过程中，结实率较高的材料染色体异常现象较少而结实率较低的材料染色体异常现象较严重。在杂种F1代中，二价体的比例要低于其相应的恢复系亲本，同样的，单价体，三价体和多价体的比例相比其恢复系亲本也偏低。然而，在减数分裂MI，杂种F1代中四价体的比例要显著高于其恢复系亲本。在中期I，每细胞单价体的比例和花粉育性呈现出极高的负相关(-0.996)，当单价体数目升高时，花粉育性下降。其次是每细胞三价体的比例(-0.987)，之后则是每细胞多价体的比例与花粉育性的负相关(-0.948)。但是统计分析表明，二价体和四价体的比例对花粉育性和结实率没有显著影响。这一结果表明出了花粉育性和细胞减数分裂行为的相关性，同源四倍体的减数分裂行为为筛选高结实率的同源四倍体种系提供了理论依据。 突变体是遗传学研究的基本材料。利用突变体克隆水稻基因,并进而研究基因的生物学功能是水稻功能基因组学的重要研究内容。本课题组在多年的四倍体水稻育种研究中已获得多个低直链淀粉含量突变体，其中一些突变体在直链淀粉含量下降的同时，胚乳外观也发生了显著改变，呈半透明或不透明。同源四倍体水稻突变株D4063-1直链淀粉含量比来源二倍体明恢63下降一半,即其直链淀粉含量为5.23%。为研究其直链淀粉含量下降的原因，我们根据普通水稻Wx基因设计引物，扩增测序获得了D4063-1Wx基因的全序列，与已报道Wx基因进行比对分析；同源四倍体水稻D4063-1Wx基因最显著变化为在外显子序列中发生了碱基缺失，导致移码突变，在第9外显子终止密码子提前出现。D4063-1Wx基因碱基位点的变化还导致了其序列上的酶切位点的变化,对常用限制性内切酶位点分析分析结果表明同源四倍体水稻相对于籼稻和粳稻多了2个sph1酶切位点，相对于粳稻减少了6个Acc1，增加了4个Xba1，1个Xho1，1个Pst1和1个Sal1酶切位点。聚类分析表明D4063-1Wx基因序列与籼稻亲源关系较近，由此推测D4063-1Wx基因来源于籼稻的Wxa基因型。另外，根据D4063-1Wx基因的碱基差异，我们推测D4063-1Wx基因外显子碱基变化导致的RNA加工障碍是其直链淀粉降低的主要原因，并可能与其米饭较软等品质相关。本文还根据D4063-1和籼稻、粳稻的序列差异并根据D4063-1在该片段上的特征序列位点设计了用于识别D4063-1的寡核苷酸片段,并作为PCR反应的引物命名为AUT4063-1,将该引物与我们设计的扩增普通籼稻、粳稻的Wx基因引物F5配合使用建立了识别D4063-1的显性和共显性两种检测方式的分子标记,为快速、准确的鉴别低直链淀粉的D4063-1创造了条件。 研究同源四倍体水稻基因组的遗传差异，探索同源四倍体水稻的遗传规律，研究分裂期染色体行为特征与遗传性状稳定性的关系,旨在揭示四倍体水稻中同源染色体配对能力的遗传差异，为进一步选育多倍体水稻品种并将其应用于生产提供理论依据。 Autotetraploid rice (2N=4X=48, AAAA) is a new germplasm developed from diploid rice (2N=2X=24, AA) through chromosomes doubling with colchicines and is an excellent resource for desirable resistance genes to the pathogens and high protein content. Therefore, heterosis utilization on polyploidy is becoming a new strategy in rice breeding. At present, the main research on autotetraploid rice centralizes in China. Breeding effort has been made to improve autotetraploid rice genetically, however, the progresses are limited due to higher degree of divergence between hybrid sterility and polygenic nature. But to date, almost nothing is reported about the genetic diversity, original and genetic background of autotetraploid rice. Despite several reports on cytological analysis of the mechanisms of low seed set in autotetraploid rice still the results are inconclusive due to lack the statistical evaluation. Therefore, the study on the mechanisms of low seed set in autotetraploid is a priority for rice breeding. Microsatellites or simple sequence repeats (SSRs) are the widely used marker for estimating genetic diversity in many species, including wild, weedy, and cultivated rice. In our research, genetic diversity and population genetic structure of autotetraploid and diploid populations collected from Chengdu Institute of Biology, Chinese Academy of Sciences were studied based on 36 microsatellite loci. For the total of 50 varieties, a moderate to high level of genetic diversity was observed at population levels with the number of alleles per locus (Ae) ranging from 2 to 6 (mean 3.028) and PIC ranging from 0.04 to 0.76 (mean 0.366). The expected heterozygosity (He) varied from 0.04 to 0.76 with the mean of 0.370 and Shannon’s index (I) ranging from 0.098 to 1.613 (mean 0.649). The autotetraploid populations showed a slightly higher level of effective alleles, the expected heterozygosity and Shannon’s index than that of diploid populations. Rare alleles were observed at most of the SSR loci in one or more of the 50 accessions and core fingerprint database of the autotetraploid and diploid rice was constructed. The F-statistics showed that genetic variability mainly existed among autotetraploid populations rather than among diploid populations (Fst=0.066). Cluster analysis of the 50 accessions showed four major groups. Group I contained all of the autotetraploid and diploid indica maintainer lines and a autotetraploid and its original diploid indica male sterile lines. Groups II contained only original of IR accessions. Group III was more diverse than either group II or IV and comprised of both autotetraploid and diploid indica restoring lines. Group IV included japonica cluster of the autotetraploid and diploid rices. Furthermore, genetic differences at the single-locus and two-locus levels, as well as components due to allelic and gametic differentiation, were revealed between autotetraploid and diploid varieties. This analysis indicated that the gene pools of diploid and autotetraploid rice are somewhat dissimilar, which made a variation that distinguishes autotetraploid from diploid rices. Using this variation, we can breed new autotetraploid varieties with some new important agricultural characters but the diploid rice has not. Cytogenetic characteristics in restorer lines DTP-4, DMinghui63 and maintainer line D46B of autotetraploid rices were studied. DTP-4, DMinghui63 and D46B showed the advantage of high seed set and biological yield. The meiotic chromosome behavior was slightly irregular in DTP-4, DMinghui63 and D46B. We observed less univalent, trivalent and multivalent at MI, but more bivalent and quadrivalent were observed. The most frequent chromosome configurations were 12II 6IVand 10II 7IV in restorer and maintainer lines, respectively. The quadrivalent frequency of DTP-4 and Dminghui63 at metaphase(MI) was respectively 2.00/PMC and 2.26/PMC. However that frequency of D46B was 6.00/PMC, which was greatly significantly higher than DTP-4 and Dminghui63. That indicates the maintainer D46B has better chromosome pairing capability in metaphase (MI). The frequency of lagging chromosomes of the maintainer D46B at anaphaseI (AI) was 10.62%, which was significantly lower than that of DTP-4(19.44%) and Dminghui63(23.14%) and nearly reaching the level of diploid CK(7.30%). In telophaseI (TI) maintainer D46B showed lower frequency of microkernel at TI and lower frequency of abnormal spores at telophaseII(TII). We also studied pollen fertility, seed set and cytogenetic characteristics of restorer lines and F1 hybrids of autotetraploid rice. DTP-4, DMinghui63, D46A´DTP-4 and D46A´DMinghui63 showed significantly higher pollen fertility and seed set than DShixiang and D46A´DShixiang. Pairing configurations in PMC of DTP-4, DMinghui63, DShixiang, D46A´DTP-4, D46A´DMinghui63 and D46A´DShixiang were 0.05 I+19.96 II (9.89 rod+10.07 ring)+0.01 III+2.20 IV, 0.11 I+19.17 II (8.90 rod+10.37 ring)+0.09 III+2.26 IV+0.01 VI, 1.33 I+9.46 II (4.50 rod+4.96 ring)+0.44 III+6.02 IV+0.09 VI+0.09 VIII, 0.02 I+14.36 II (6.44 rod+7.91 ring)+0.01 III+4.80 IV+0.01V III, 0.06 I+17.67 II (11.01 rod+6.67 ring)+0.06 III+3.10 IV+0.01 VI and 1.11 I+11.31 II (5.80 rod+5.51 ring)+0.41 III+5.63 IV+0.03 VI+0.03 VIII, respectively. Configuration 16 II+4 IV and 12 II+6 IV occurred in the highest frequency among the autotetraploid restorers and hybrids. Meiotic chromosome behaviors were less abnormal in the tetraploids with high seed set than those with low seed set. The hybrids had fewer frequencies of bivalents, univalents, trivalents and multivalents than the restorers, but higher frequency of quatrivalents than the restorers at MI. The frequency of univalents at M1 had the most impact on pollen fertility and seed set, i.e., pollen fertility decreased with the increase of univalents. The secondary impact factors were trivalents and multivalents, and bivalents and quatrivalents had no effect on pollen fertility and seed set. The correlative relationship between pollen fertility and cytogenetic behaviors could be utilized to improve seed set in autotetraploidy breeding. The amylose content of autotetraploid indica mutant Rice D4063-1 dropped by half than diploid Minghui 63, that is, its amylose content of 5.23%.The whole sequence of Waxy gene of D4063-1 is amplified and sequenced. And the discrepancy of bases is found comparing to the reported Waxy gene. The Waxy gene of autotetraploid Rice D4063-1 had a base deletion in exon sequence, which resulted frameshift mutation in exon 9 and termination codon occur early. The mutation of Wx also led to the change of some common restriction endonuclease sites. Results showed compared to indica and japonica, D4063-1 had two adding sph1 sites. Compared to japonica, D4063-1 had six decreasing Acc1, a adding Xho1, Pst1 and Sal1 restriction sites. Phylogeny analysis shows that the DNA sequence of Waxy gene of D4063-1 is closer to Indica, and we suppose that the Waxy gene of D4063-1 is origin from genotype Wxa. In addition, according to the base differences of Wx in D4063-1, we deduce that RNA processing obstacle led by base change of intron is the main cause to low the amylose content, and related to phenotype of its soft rice. Based on analysis of fragments of D4063-1, indica and japonica and according to the special point of the three species, primers as markers-AUT4063-I were designed for distinguishing the D4063-1 from other rice. Combining with primer pair F5, dominant and codominant ways were established for discriminating them., rapid and correct identification of D4063-1 from other rice could be done. The genetic analysis is important to ensure the original of autotetraploid rice, for maintaining the “distinctiveness” of autotetraploid varieties, and to differentiate between the various genetic background of autotetraploid rice. The autotetraploid breeding will benefit from detailed analysis of genetic diversity in the germplasm collections. Further investigation on mechanisms of meiotic stability should benefit polyploid breeding. These findings demonstrated opportunity to improve meiotic abnormalities as well as grain fertilities in autotetraploid rice.

自养氨氧化混合种群特性和共存机理初步研究

组特殊自养氨氧化混合种群，表现：无机环境种群生长迅速、生物量高；在一个完全无机的自养生长环境中，不仅保持高氨氧化速率，并出现丰富的异养微生物种群；该种群置于异养、厌氧环境中，迅速表现出产氢特征。对于这样一个特殊的生态体系，研究其共生机理，以及联接这些种群之间的碳源和能源问题，将具有非常重要意义。我们拟从种群特征、细胞表面分泌产物、游离体系产物多糖、蛋白和脂肪酸方面开展研究。 第一部分，自养氨氧化混合种群的基本特征。采用氨氧化培养基，进行种群氨氧化特征研究；采用扫描电镜观察自养混合种群的微观特征；沉降、离心去除微生物种群，分析水相中的总有机碳、糖类等物质；利用LB培养基进行种群的分离、纯化，并采用DGGE手段对微生物种群结构进行分析。结果表明，接入菌种后（2/5000（V/V）），培养液中氨（200mg/L）在3－5天内快速降解；亚硝酸盐与氨氮变化呈负相关趋势，仅有少量硝酸盐含量（< 30mg/L）。氨氧化种群的生物量增长与氨氧化趋势一致，初始生物量7.75 mg/L(蛋白含量)，3-5天后生物量快速增长，并达到最高63.06 mg/L（蛋白含量）。电镜图片显示，种群外包裹一层粘液。离心除去菌体后，检测培养液总有机碳和糖的含量，同样表现出与生物量增长相似的特征，分别由初始的3.73、2.35 mg/L，3－5内天迅速增加，并分别达到最大值35.19、27.45 mg/L。经初步分离、纯化并对纯化菌株进行测序，获得了10株异养微生物分别为布鲁氏菌科苍白杆菌属、纤维单孢菌、类芽孢菌属、黄杆菌属、无色杆菌、鞘脂单胞菌、嗜麦芽寡养单胞菌、噬氢菌属、硫红球菌、假单胞菌；DGGE显示，约有20分条离带，我们对其中的两条优势条带进行切割回收测序，鉴定为欧洲亚硝化单胞菌（Nitrosomonas eur）。 第二部分：混合种群自养-异养菌共生的可能机制。在对微生物种群特征初步分析基础上，针对胞外糖类组分可能被微生物代谢分解，我们重点对微生物细胞蛋白质与糖类进行分析。采用超声结合RIPA裂解液裂解，SDS-PAGE电泳分析混合种群总蛋白种类，并通过氨基酸分析仪及红外光谱法分析氨基酸组成及蛋白红外特征。采用超声破碎结合反复冻融对细胞样品进行处理，提取液采用醇沉、Sevage脱氮白，凝胶过滤方法脱盐和分级分离。对提取物的糖分析包括：紫外扫描，红外光谱，核磁共振，单糖组成分析；扫描电镜观察菌群破裂现象。SDS-PAGE分析结果表明：氨氧化种群不同生长阶段都显示出42kD蛋白表达量很高，d4时42kD蛋白表达已经很强，4-7d内一直持续这种过量表达，直到d8后表达开始减弱。说明42kD蛋白可能与氨氧化密切相关。红外光谱分析显示：细胞提取物的特征峰分布在3427.42cm-1、1718.18 cm-1和1681.72 cm-1、1160.07和1086.74 cm-1，分别对应为OH、 C=O、C－O－C基团，表明具有蛋白的典型特征；氨基酸分析显示蛋白中的Gly,Asp,Ala,Glu含量相对较高。 提取物中胞外多糖分离谱图得到不均一组分，共得到6个收集峰；紫外扫描在201－213 nm处有多糖吸收峰，同样表明多糖成分不均一性；多糖红外光谱特征峰主要分别在3400.49 cm-1、2920.28 cm-1、1154.54和1087.52 cm-1，对应OH、－CH2－ or CH 、C－O－H or C－O－C等多糖特征基团；多糖提取物核磁共振1H d4.3～5.9之间出现强吸收峰，这是1H中，多糖存在的明显证据，1H NMR中，其中O-乙酰基的甲基上的氢信号为d1.1～1.3之间。糖肟全苯甲酸酯衍生物的HPLC测定中，得到单一的单糖峰，由于时间问题，还未进行更深入的试验；电镜图片显示，种群中的细胞有大量的破裂现象。 实验表明，自养氨氧化混合种群显示出快速的氨氧化速率，氨氧化过程生物量和有机质的增加明显。微生物种群包裹粘液层，并分离纯化出大量的异养菌；去除菌体后的游离培养液中存在有机质（包括多糖）说明无机自养生长体系中存在异养菌生长、繁殖的二次碳源；细胞提取物中蛋白条带数目多、种类丰富；细胞多糖提取物具有明显的多糖特征，以及单糖的存在。结合种群的显微特征和游离体系中的有机质的检测结果，我们认为，无机自养生长体系中，种群细胞生长过程中发生的破裂现象可能是导致大量的蛋白、多糖释放到游离胞外，并成为其他异养菌生长的碳源和氮源。这可能是自养体系中，大量异养菌共生的可能机制，至于是什么原因引起种群生长过程中产生的破裂现象，还有待下一步深入研究。 A group of mixed autotrophic ammonia oxidizing populations, having much biological characteristic tested by concerned personnel for pilot test: Performed rapid population growth and obtained high biomass in inorganic environment; Not only maintained a high rate of ammoxidation, promoted a wealth of heterotrophic microbial populations growth in a totally inorganic and autotrophic growth environment; Placed in heterotrophic and anaerobic environment,had the performance characteristics that could rapidly produce hydrogen.For such a special ecological system, Study its symbiotic mechanism and the connection between these populations of carbon and energy issues, will have a very important significance. We intended from the characteristics of the population, the secretion product of cell surface, free substance in the liquid medium like polysaccharide, protein and fatty acids carrying out research. Part I: The basic features of mixed autotrophic ammonia oxidizing populations . Use inorganic liquid medium, processed study for ammonia oxidation characteristics of the population; we used scanning electron microscopy to get micro-features of autotrophic ammonia oxidizing populations .The medium was carried out settlement and centrifugal then removed the microbial populations, after all of that we analysis the water phase for total organic carbon(TOC), carbohydrate and other substances; Solid ammonia oxidizing medium was adopted to separation and purification of population, DGGE means was for structure analysis of microbial population. The results showed that after the inoculum of bacteria (2 / 5000 (V / V)), ammonia in the culture medium (200 mg / L) was rapid degradation in 3-5 days; ammonia and nitrite have the negative correlation between changes in the trend, then only a small amount of nitrate content (<30mg / L). The biomass growth of ammoxidation population in line with the trend of ammonia oxidation, the initial volume of it was 7.75 mg / L (protein content), in 3-5 days upto 63.06 mg / L (protein content). Electron microscope image showed, the populations were wrapped in a layer of mucus, including the a large number ruptted micorbe , Centrifuge to remove bacteria, then detected the medium for total organic carbon and sugar content, result took on the same characteristics with biomass growth, that were from the initial 3.73、2.35 mg / L respectively, in 3-6 days achieved rapid increase in the maximum to 35.19、27.45 mg / L respectively. After initial separation、 purification ,then processed sequencing to strains purified and got the result that there were 10 heterotrophic microorganisms : Brucella Branch pale bacillus, Cellu lomonas, Bacillus species category, a Flavobacterium, colorless Bacteria, Aeromonas sheath fat, little support maltophilia Aeromonas, macrophages species hydrogen, sulphur-MI, Pseudomonas bacteria spores; DGGE display, there were 20 separation bands approximately. Part II: Mixed populations that autotrophic - heterotrophic bacteria symbiotic mechanism. On the basis of preliminary analysis of microbial population characteristics, aiming at extracellular carbohydrate components might be decomposition by microbial, we focused on microbial cell protein and carbohydrate analysis. Using ultrasound combined with RIPA lysis cracking the cells, SDS-PAGE electrophoresis analysis the total protein species of the population, and through the amino acid analyzer studied the compositions of amino acid and infrared spectroscopy analysis of a protein infrared characteristics. Using ultrasound combined with repeatedly freezing and thawing to treated the cell sample, then took the means that alcohol precipitation, deproteinization by Sevage, gel filtration aimed at desalination and grade separation to deal with the lysates . The extraction of sugar analysis included: UV scanning, IR, NMR, single-sugar composition analysis. SDS-PAGE analysis showed that: 42 kD protein expression was very high at different growth stages of mixed autotrophic ammonia oxidizing populations , on the fourth day, 42 kD protein expression had been very strong, 4-7d, it had continued this excessive expression, then started to weaken after 7 days. 42 kD protein that might be closely associated with ammonia oxidation. Infrared spectral analysis showed that: cell extracts with the characteristic that the peak distribution in 3427.42 cm-1、1718.18 cm-1 and 1681.72 cm-1、1160.07 cm-1 and 1086.74 cm-1 corresponding to OH、C = O、C-O-C Groups which had the typical characteristics of protein; and analysis showed that amino acids including Gly, Asp, Ala, Glu ,the content in the protein is relatively high. Exopolysaccharide in the extracts had the separation map that it was uneven, received a total of six collection peaks by the detection mode of phenol-sulphruic acid method ; ultraviolet scan in the 201-213 nm department had polysaccharide absorbing peak, the same ingredients that polysaccharide heterogeneity; infrared polysaccharide spectral characteristics of the main peak at 3400.49 cm-1, 2920.28 cm-1, 1154.54 and 1087.52 cm-1, corresponding OH,-CH2-or CH, C-O-H or C-O-C;and other characteristics of polysaccharide group; 1H NMR of polysaccharide extract appeared absorption peak between d4.3 ~5.9, which is the apparent evidence of polysaccharide, In 1H NMR, the hydrogen signal of one of O-acetyl was between 1.1 to 1.3. The determination of Sugar oxime whole benzoate derivatives by HPLC, there was a single-sugar peak, as a matter of time, yet more in-depth test. Summary: Mixed autotrophic ammonia oxidizing populations show us that it had the ability in ammonia oxidizing and it was great, organic matter and biomass increased significantly in the process of ammonia oxidation. Microbial populations was wrapped up slime layer, the phenomenon of cell breakdown obviously, and there were a lot of separation and purification of the heterotrophic bacteria; a lot of organic matter (including polysaccharides)remined in the medium that removal of cell indicated the inorganic system existed secondary carbon sources that could be used by the heterotrophic bacteria ; there were a large number proteins bands of cell extract, rich variety; cell extracts of polysaccharide had obvious characteristics of polysaccharide, and the existence evidence of single-sugar. Combined population of microscopic characteristics and free of organic matter in the test results, we believe that the health of inorganic system, population growth occurred in the course of the breakdown of the phenomenon is likely to lead to a lot of protein and polysaccharide released into the extracellular free, And other heterotrophic bacteria use them to the growth as carbon and nitrogen. This may be autotrophic system, the large number of heterotrophic bacteria symbiotic mechanism.

Defensive and Sensory Chemical Ecology of Brown Algae

The ecological interaction of brown algae are important as these macroalgae are common and often keystone members in many benthic marine communities.This review highlights their chemical interactions,particularly with potential herbivores,but also with fouling oranganisms,with potential pathogens,with each other as gametes,and with their microenvironments when they are spores.Phlorotannins,which are phenolic compounds unique to brown algae,have been studied hesvily in many of these respects and sre highlightes here.This includes recent controversy about their roles as defences against herbivory,as well as new understanding of their roles in primary cellular functions that may,in many instances,be more important than ,and which at least have to be considered in convert with,any possible ecological functions.Brown algae have also been useful models for testing theoties about the evolution of and ecological constraints on chemical defence.Furthermore,their mocroscopic motile gametes and spores have the ability to react to their chemical environments behavirourally.

The Development of a Marine Natural Product-based Antifouling Paint

Problems with tin and copper antifouling compounds have highlighted the need to develop new environmentally friendly antifouling coatings. Bacteria isolated from living surfaces in the marine environment are a promising source of natural antifouling compounds. Four isolates were used to produce extracts that were formulated into ten waterbased paints. All but one of the paints showed activity against a test panel of fouling bacteria. Five of the paints were further tested for their ability to inhibit the settlement of barnacle larvae, Balanus amphitrite, and algal spores of Ulva lactuca, and for their ability to inhibit the growth of U. lactuca. Two paints caused a significant decrease in the number of settled barnacles. One paint containing extract of Pseudomonas sp. strain NUDMB50-11, showed excellent activity in all assays. The antifouling chemicals responsible for the activity of the extract were isolated, using bioassay guided fractionation, and their chemical structures determined.