西洋参体细胞胚胎发生的研究

以西洋参(Panax quinquefolium L.)为材料，从胚、胚乳、多年生主根等外植体诱导筛选得到多种胚性及非胚性愈伤组织，胚来源的胚性愈伤组织经用附加1.0ppm 2,4-D的MS培养基继代保持三年后仍具旺盛的体细胞胚胎发生能力，以胚性愈伤组织建立的液体培养系统可得到大量游离的胚状体，将胚状体包埋制成的人工种子能在附加0.1ppm NAA和1.0ppm GA_3的B_s培养基上萌发形成根、茎、叶健全的再生植株，并且移栽成活。松软型根愈伤组织以过长期继代培养后，也得到了胚状体发生。比较了多种培养因素对体细胞胚胎发生和愈伤组织的影响，其中外植体来源、基本培养基的无机盐组成以及生长素是决定愈伤组织形态结构类型和体细胞胚胎发生能力的主要因素。胚乳愈伤组织、质密型根愈伤组织以及来源于胚的松软型非胚性愈伤组织在多种诱导条件下均未能发生胚状体。 从松软型的胚或根愈伤组织均容易游离大量原生质体，用悬浮培养物游离原生质体的得率更高，从早期胚状体也可酶解得到可用于培养量的原生质体。原生质体体积均很小，培养中的行为也相似，有些形成细胞壁，细胞变形，个别进行细胞分裂，形成少数细胞团，但未形成愈伤组织;有些原生质体仍保持球形，体积剧裂膨大几十倍，小液泡汇聚成大液泡，有些液泡中积累紫红色素。 用石蜡切片、半薄切片、透射电镜和扫描电镜等方法对各种愈伤组织的内部结构及外部形态进行了详细观察，发现胚状体起源于愈伤组织的表皮或表皮下层细胞，有单个发生和成丛发生两种发生方式，不同愈伤组织细胞的显微及超微结构各具特色。胚乳愈伤组织、松软型根愈作组织以及胚状体的细胞虽然都具有某些胚性细胞结构特征，例如细胞体积小、细胞核大、细胞质浓厚、细胞器丰富等，但它们并不具有现实的体细胞胚胎发生能力。发生胚状体的细胞含大量多核糖体和内质网片段，小液泡数量少且形成多泡复合体，细胞壁也由于在组织中所外的位置不同而有厚薄两种类型之分，细胞常形成胚性细胞复合体，位于愈伤组织的外围，边缘可分化为许多小细胞团。胚性细胞复合体的表面质密平整，有许多丝状物和颗粒，细胞轮廓不清，但有显著突出的小细胞，细胞表面具沟脊;而其它各型愈伤组织的表面均为球形或半球形的大型细胞，表面仅具细小的纹理、凸起或颗粒。 胚来源的胚性愈伤组织的总蛋白组成与来源相同，但形态结构截然不同的非胚性愈作组织的总蛋白组成差异显著，后者与形态结构特征相似的根本源公软型愈伤组织具有相似的蛋白质电泳带型，但多一条33KD的蛋白带;培养基中去掉2,4-D后，松软型根愈伤组织的蛋白质组成发生轻微变化。等电聚焦和SDS聚丙烯酰胺凝胶双向电泳揭示出在单向电泳扫描上呈现最高峰的17KD蛋白质在胚性愈伤组织中随等电点的不同而分离，在其它三种构软型愈伤组织中则仅集中在等电点为5.9处，为一个大而染色深的点。 同样培养基上培养的形态结构不同的愈伤组织所含元素的量不同，说明细胞对无机盐的吸收是有选择性的，不同类型的愈伤组织可积累不同的元素。培养基中的元素组成情况在愈伤组织中也有一定反映，在含钠、钾元素较多的B_5培养基上生长的愈伤组织中这两种元素的含量也较高，培养基的离子胁迫作用和细胞对离子一定程度的被动吸收会影响细胞的代谢方式，从而导致细胞类型的分化，基本培养基对愈伤组织类型的转变及体细胞胚胎发生能力的影响可能正是通过影响细胞的离子吸收、代谢平衡而实现。

An ecological characterization of the marine resources of Vieques, Puerto Rico. Part I: Historical data synthesis

From the 1940s until 2003, portions of the island of Vieques, a municipality within the Commonwealth of Puerto Rico, were used by the US Navy as a base and training facility, resulting in development and zoning history that differ in comparison to other Caribbean islands. The majority of former Navy lands are now under the jurisdiction of the Department of the Interior’s Fish and Wildlife Service as a National Wildlife Refuge, while a smaller percentage of land was transferred to the Vieques municipality and the Puerto Rico Conservation Trust. An analysis of the distribution and status of the marine resources is timely in light of the recent land transfer, increases in development and tourism, and potential changes in marine zoning around the island. To meet this need, NOAA’s Biogeography Branch, in cooperation with the Office of Response and Restoration and other local and regional partners, conducted Part I of an ecological characterization to integrate historical data and research into a synthesis report. The overall objective of this report is to provide resource managers and residents a comprehensive characterization of the marine resources of Vieques to support research, monitoring, and management. For example, knowledge of the spatial distribution of physical features, habitats, and biological communities is necessary to make an informed decision of the establishment and placement of a marine protected area (MPA). The report is divided into chapters based on the physical environment (e.g., climate, geology, bathymetry), habitat types (e.g., reefs and hardbottom, seagrasses, mangroves) and major faunal groups (e.g. fish, turtles, birds). Each section includes five subsections: an overview, description of the relevant literature, methods of analysis, information on the distribution, status and trends of the particular resource, and a discussion of ecological linkages with other components of the Vieques marine ecosystem and surrounding environment. The physical environment of Vieques is similar to other islands within the Greater Antilles chain, with some distinctions. The warm, tropical climate of Vieques, mediated by the northeasterly trade winds, is characterized by a dry season (December-April) and a rainy season (May-November), the latter of which is characterized by the occasional passage of tropical cyclones. Compared to mainland Puerto Rico, Vieques is characterized by lower elevation, less annual precipitation, and higher average temperatures. The amount of annual precipitation also varies spatially within Vieques, with the western portion of the island receiving higher amounts of rainfall than further east. While the North Equatorial Current dominates the circulation pattern in the Greater Antilles region, small scale current patterns specific to Vieques are not as well characterized. These physical processes are important factors mitigating the distribution and composition of marine benthic habitats around Vieques. In general, the topography of Vieques is characterized by rolling hills. Mt. Pirata, the tallest point at 301 m, is located near the southwest coast. In the absence of island wide sedimentation measurements, information on land cover, slope, precipitation, and soil type were used to estimate relative erosion potential and sediment delivery for each watershed. While slope and precipitation amount are the primary driving factors controlling runoff, land use practices such as urban development, military activity, road construction, and agriculture can increase the delivery of pollution and sediments to coastal waters. Due to the recent land transfer, increased development and tourism is expected, which may result in changes in the input of sediments to the coastal environment.

细胞分裂素的测定及其转基因植物的基因表达和调控的研究

细胞分裂素是一类重要的植物激素，它参与调节许多植物的生命活动过程。本文从几个方面研究了细胞分裂素的作用。 在细胞分裂素的活性测定中，通过改进尾穗蔸苋红素合成法建立了一种简便、准确的生物试法，同时还建立了根据物理化学和免疫学原理而测定细胞分裂素的HPLC和ELISA方法，使得细胞分裂素的定量更加准确。经过对上述三种方法的相互验证实验表明，同时采用二种方法可以保证细胞分裂素分析的准确和可靠。 细胞分裂素可以促进黄瓜子叶的扩张。利用离体黄瓜子叶，分析BA诱发其扩张与子叶内源细胞分裂素之间的关系，实验证明，BA能促进玉米素及其核苷的迅速积累，进而诱发子叶的扩张。上述结果还表明，黄瓜子叶可能具有合成细胞分裂素的能力。 荸荠球茎是一种贮藏器官，但实验测定发现其中含有细胞分裂素的生理活性形式——异戊烯基腺嘌呤核苷(iPA)，而且合成它的前体腺嘌呤的含量也十分丰富，考虑到球茎与种子的类似之处，推测它可能做为合成细胞分裂素的一个源，而且其合成途径可能有别于植物其它组织。 农杆菌中的异戊烯基转移酶(ipt)基因是负责细胞分裂素生物合成的关键基因。将ipt基因克隆后对其启动子进行了改造，分别构建了如下三种基因：(1) ipt启动子+ipt编码区和3，区(ipt)，(2)磷酸核酮糖羧化酶小亚基启动子SSU 301+ipt编码区和3，区(SSU -ipt)，(3)豌豆种子特异性启动子viciln+ipt编码区和3，区(vic-ipt)。上述三种基因经农杆菌介导转化烟草，获得了16株再生植株，经Southern杂交证明其中15株的基因组上含有正常整合的ipt基因。Northern杂交表明有13株转基因烟草中的ipt基因能转录出大小正常的ipt mRNA并促进了细胞分裂素的生物合成。 实验表明，转基因烟草中ipt基因的表达受到多种因素的调控。首先启动子决定了ipt基因的表达模式，SSU -ipt基因的表达受光的诱导，黑暗中这种基因的转录完全停止，而vic-ipt基因的表达是种子特异性的，它不在烟草营养生长器官如根、茎、叶和愈伤组织中表达。第二，生长素能降低ipt基因的表达活性。第三，在整体植物的根中，存在某些反式因子，能够控制ipt基因的过量表达，这其中可能涉及到细胞内的蛋白因子、基因的甲基化作用及细胞分裂素的反馈调节等。 vic-ipt基因在烟草种子中的特异性表达导致种子内形成了一个细胞分裂素合成的源(source)。对种子中营养物质积累的研究表明，ipt基因的表达促进了种子干物质的积累，其中作用最明显的是增加种子内蛋白质的合成。转入vic-ipt基因后的烟草种子其萌发率没有显著变化，但幼苗的生长速率明显加快，这表明细胞分裂素能调节植株的生长。 通过Northern杂交检测转基因烟草中基因表达的调控，实验证明，ipt基因的表达明显抑制一组植物病理相关蛋白(PR)基因的转录活性，这组基因编码：几丁质酶，β-1，3一葡萄糖苷酶，伸展蛋白和渗调蛋白。对这些调控作用的生理学意义还有待进一步探索。 上述结果表明，在高等植物中，除了传统上认为根是合成细胞分裂素的部位之外，其它组织和器官也具有合成细胞分裂素的能力，其中合成能力最强的是一些离体组织和贮藏器官。农杆菌中的细胞分裂素生物合成基因(ipt)能够在高等植物的基因组中正常的整合和表达，并受到植物体内生理、发育等多种因素的调控，而与整体植物的正常生理过程协调一致。ipt基因的表达还能够调节植物体的生长和发育，包括种子发育时营养物质的积累、幼苗的生长和某些相关基因的表达。对上述问题的深入研究,必将促进细胞分裂素及其相关生理学和发育学研究的进展。

PEFCSH 的建立及在基因组特异序列克隆上的应用

基因组特异序列是跟踪外源染色质、鉴定易位系的特异探针。本文介绍一种带反馈控制的PCR增效减法杂交（PEFCSH），证明可高效克隆基因组特异序列。 带PCR接头的黑麦DNA片段与固定化的小麦ssDNA杂交，同源的片段将被吸附。用PCR扩增吸附的DNA，可监测杂交液中与小麦同源的DNA，确定是否还要再杂交。5轮连续杂交后，杂交液中的DNA几乎全为黑麦特异DNA，纯化后，用PCR扩增到方便操作的数量。经检测，PEFCSH片段99%为黑麦基因组特异性序列，富集度超过230倍。 PEFCSH片段克隆后检测：插入片段在120bp～2000bp，峰值250bp左右;306个克隆中301个显黑麦特异性，表明了PEFCSH的高效性。Tomita等曾用普通减法杂交富集黑麦特异序列，所得克隆只有6.3%为黑麦特异。 与数据库对比，分离片段有的为新序列，更多的与已知的黑麦特异重复序列同源。用其中一条作探针进行Southern杂交，小麦不显带，黑麦显阶梯型带，说明它是特异性串联重复序列。 PEFCSH有如下特点：1. 实时监测杂交液中非特异DNA，首次引入反馈控制，确保杂交达到预期效果。2. 用PCR制备Tester只需少量样品就可以分离特异序列。3. 采用固相减法杂交，大大简化Test与Driver的分离。4. 用PCR克服常规减法杂交操作性差的弱点。5. 富集特异单链和双链DNA，减少特异序列丢失。6. 适用于大多数分离两组相关核酸中的差异成分。

Reproductive dynamics of female spotted seatrout (Cynoscion nebulosus) in South Carolina

We describe reproductive dynamics of female spotted seatrout (Cynoscion nebulosus) in South Carolina (SC). Batch fecundity (BF), spawning frequency (SF), relative fecundity (RF), and annual fecundity (AF) for age classes 1−3 were estimated during the spawning seasons of 1998, 1999, and 2000. Based on histological evidence, spawning of spotted seatrout in SC was determined to take place from late April through early September. Size at first maturity was 248 mm total length (TL); 50% and 100% maturity occurred at 268 mm and 301 mm TL, respectively. Batch fecundity estimates from counts of oocytes in final maturation varied significantly among year classes. One-year-old spotted seatrout spawned an average of 145,452 oocytes per batch, whereas fish aged 2 and 3 had a mean BF of 291,123 and 529,976 oocytes, respectively. We determined monthly SF from the inverse of the proportion of ovaries with postovulatory follicles (POF) less than 24 hours old among mature and developing females. Overall, spotted seatrout spawned every 4.4 days, an average of 28 times during the season. A chronology of POF atresia for water temperature >25°C is presented. Length, weight (ovary-free), and age explained 67%, 65%, and 58% of the variability in BF, respectively. Neither RF (number of oocytes/g ovary-free weight) nor oocyte diameter varied significantly with age. However, RF was significantly greater and oocyte diameter was smaller at the end of the spawning season. Annual fecundity estimates were approximately 3.2, 9.5, and 17.6 million oocytes for each age class, respectively. Spotted seatrout ages 1−3 contributed an average of 29%, 39%, and 21% to the overall reproductive effort according to the relative abundance of each age class. Ages 4 and 5 contributed 7% and 4%, respectively, according to predicted AF values.

Age and size composition, growth rate, reproductive biology, and habitats of the West Australian dhufish (Glaucosoma hebraicum) and their relevance to the management of this species

Samples of the commercially and recreationally important West Australian dhufish (Glaucosoma hebraicum) were obtained from the lower west coast of Australia by a variety of methods. Fish <300 mm TL were caught over flat, hard substrata and low-lying limestone reefs, whereas larger fish were caught over larger limestone and coral reef formations. Maximum total lengths, weights, and ages were 981 mm, 15.3 kg, and 39 years, respectively, for females and 1120 mm, 23.2 kg, and 41 years, respectively, for males. The von Bertalanffy growth curves for females and males were significantly different. The values for L∞, k, and t0 in the von Bertalanffy growth equations were 929 mm, 0.111/year, and –0.141 years, respectively, for females, and 1025 mm, 0.111/year, and –0.052 years, respectively, for males. Preliminary estimates of total mortality indicated that G. hebraicum is now subjected to a level of fishing pressure that must be of concern to fishery managers. Glaucosoma hebraicum, which spawns between November and April and predominantly between December and March, breeds at a wide range of depths and is a multiple spawner. The L50’s for females and males at first maturity, i.e. 301 and 320 mm, respectively, were attained by about the end of the third year of life and are well below the minimum legal length (MLL) of 500 mm. Because females and males did not reach the MLL until the end of their seventh and sixth years of life, respectively, they would have had, on average, the opportunity of spawning during four and three spawning seasons, respectively, before they reached the MLL. However, because G. hebraicum caught in water depths >40 m typically die upon release, a MLL is of limited use for conserving this species. Alternative approaches, such as restricting fishing activity in highly fished areas, reducing daily bag limits for recreational fishermen, introducing quotas or revising specific details of certain commercial hand-line licences (or doing both) are more likely to provide effective conservation measures.