陆地棉抗虫基因工程研究

以陆地棉（Gossypium hirsutum L.）栽培品种新陆早4号、系550、冀资492、衡无89-30、邯93-2、冀资123等为材料，进行了组织培养及植株再生研究，建立了一套陆地棉体细胞植株再生速成体系。通过调整激素种类与比例以及改善培养条件，降低了畸形胚发生频率（从80%降为41%），并可将畸形苗转化为正常苗（转化率约为78%）;通过水培和嫁接，结合试管扦插、扩繁技术，解决了棉花生根及移栽难题，为农杆菌介导法转化棉花奠定了基础。 用绿色荧光蛋白基因（gfp）作为报告基因，构建了pBGb1m（含Bt和gfp二价基因）、pBGbf（含Bt-gfp融合基因）和pBGbfg（含Bt-gfp融合基因和gna基因）等三种植物表达载体。通过农杆菌介导法转化烟草，转基因再生植株经过荧光、虫试、PCR、Southern blot和Western blot等检测，表明三种植物表达载体能够在转基因植物中有效表达，同时，绿色荧光蛋白（GFP）的检测表现出了简便、经济、快速、可靠等优点，为大量棉花转基因苗的检测提供了一种有效方法。 采用花粉管通道法将携带细胞间隙定位信号肽的Bt基因的pBin438-S1m质粒导入棉花品种冀资492，经过田间卡那霉素筛选、虫试、PCR、PCR-Southern blot和Southern blot检测，证明Bt基因已整合至棉花基因组中，而且可能是以单拷贝形式插入。 同时，通过农杆菌介导法将三种植物表达载体（pBGb1m、pBGbf和pBGbfg）转化陆地棉栽培品种新陆早4号、冀资492、衡无89-30和邯93-2等材料，获得了大量转化再生棉株。经过PCR和PCR-Southern blot检测，转基因阳性植株为转为再生植株总数的89.45%。目前，虫试、Southern blot及Western blot正在进行之中。

棉花抗黄萎病基因工程研究和防卫反应相关基因的克隆

系统获得性抗性(Systematic acquired resistance, SAR)是植物抵御病原菌侵染的最有效手段，利用基因工程技术导入SAR信号发生过程中的关键基因后，植物的SAR基因表达量提高并且对病原菌侵染的反应速度加快，因此植物的抗病性得以增强，与传统的抗病基因工程技术相比它对病原菌没有专一性，许多学者称之为广谱抗病基因工程，该领域已成为目前抗病基因工程研究的热点和前沿。 NDR1和NPR1基因在植物的SAR发生中起着重要的作用，前者功能定位在ROS(reactive oxygen species)的激活和随后的水杨酸(SA)诱导合成之间，突变株病原菌诱导后SA合成能力降低，SAR发生减弱，目前还没有对该基因进行过量表达分析的报道;后者功能定位在SAR信号转导级联反应之中的SA积累和随后的SAR基因表达之间。该突变株在病原菌侵染时不产生病程相关蛋白(PRs)，表现为感病，而对照抗病;过量表达该基因的转基因拟南芥对多种病原菌的侵染产生抗性，PR1等PRs蛋白的表达量也提高，异源表达该基因的水稻对白叶枯病的抗性也提高。本研究利用RT-PCR方法从拟南芥中克隆了这两种基因，序列分析表明拟南芥Wassilewskija生态型的NDR1基因与Columbia生态型相比，共有7处碱基不同，引起编码氨基酸变化4处，而NPR1基因与报道的Wassilewskija生态型来源的NPR1基因完全相同。 我们构建了35S启动子驱动的NDR1和NPR1基因的植物组成型高效表达载体，利用农杆菌介导法转化烟草，PCR和Southern鉴定外源基因已经整合到植物基因组中。抗病性分析显示过量表达NDR1和NPR1基因的烟草对晚疫病和赤星病的抗性都有明显提高，说明这两个基因的在其它植物中异源表达后，都能提高植物对多种病原菌的抗性。 本论文提出了利用这两个基因来培育抗黄萎病棉花的设想，一方面为解决这个“世纪性”难题积累新的资料，另一方面也为其它作物的抗病基因工程提供新的经验。利用35S启动子驱动的NDR1和NPR1基因的植物组成型表达载体分别对陆地棉品种石远321进行花粉管通道法转化。同时，还探讨了这两个基因在棉花中的共转化实验，希望它们的“协同增效”能进一步提高棉花的抗病性。对其中2001年夏天在南京注射所获得的5,000粒种子在三亚进行100 g/ml卡那霉素筛选，初步鉴定分别获得转NDR1和NPR1基因株系26和24棵，PCR进一步鉴定其中分别有12和7棵为转基因阳性，转基因频率分别为0.50％和0.27％，目前利用营养钵蘸根法对其二代进行抗枯、黄萎病鉴定，结果显示有转基因植株对枯、黄萎病的抗性都明显增强，进一步的鉴定正在进行中。2002年初海南注射分别获得转NDR1和NPR1基因以及共转化种子22,000、10,500和12,500粒种子，2002年夏在中国农科院植保所黄萎菌病圃筛选抗黄萎病单株，并利用100 g/ml卡那霉素初步筛选出了一批抗性植株，每种转基因株系随机挑选5株进行PCR鉴定，结果显示为阳性。进一步的抗黄萎病鉴定和筛选以及分子分析正在进行中。 同时，本文还探讨了病原菌诱导型启动子在广谱抗病基因工程应用的可能性。根据烟草的Pr1-a启动子已知序列设计引物，PCR扩增启动子序列后,构建病原菌诱导型NPR1基因植物表达载体，并对棉花进行转化，获得种子11,500粒，利用同上的筛选方法，获得了一致的结果，目前抗黄萎病鉴定、分子检测以及生物学分析正在进行中。 最后，鉴于抗生素标记在转基因植物的应用引起了许多“安全性”争论的事实，还构建了无筛选标记的表达载体对抗虫棉进行转化，这样在生产上可以直接获得抗虫棉抗黄萎病棉花新材料，也为其它作物抗病基因工程积累经验。 本研究还提出了一种较为有效的提取高质量棉花总RNA的方法，与原来一些棉花RNA纯化方法相比，该方法所用都为常规试剂，易于重复，质量高。并且利用获得的总RNA构建了黄萎菌激发子诱导的cDNA文库，滴度测定为1╳107pfμ/μg，插入片段大小在5 00~2 000 bp范围内。 鉴于NPR1基因研究的重要性，本研究还利用简并引物PCR技术从海岛棉和陆地棉的基因组中都分离到了NPR1基因的同源片段，大小都为208 bp，与拟南芥NPR1基因的相应部分的同源性分别为66％和65％，它们之间的同源性为87％，目前该基因的全长正在分离鉴定中。 多聚半乳糖醛酸酶抑制蛋白(PGIPs)在植物的防御反应中起着重要的作用，通过分析已知20余种pgip基因序列的保守区，设计简并引物，PCR扩增海岛棉（Gossypium barbadense）7124 cDNA文库，得到一条长561 bp的片段，序列测定后分析确认为pgip基因的一部分。根据此序列和棉花病原菌诱导的cDNA文库载体中已知部分设计RACE引物，扩增后，5’和3’RACE分别得到666bp和906 bp的片段。序列分析表明它具有完整的编码框，产物为330 aa的蛋白质。序列分析该蛋白具有10个串联的LRR(leucine-rich repeat)区，与柑桔(Citrus)和枳(Poncirus)的pgip基因的同源性分别为69.2%和68.7%。进一步PCR扩增得到该基因的全长阅读框，并且获得了相应的基因组片段，序列分析发现该基因没有内含子。这是从棉属植物中克隆的第一个pgip基因。

生物安全信息建设和转基因棉花根际土壤微生物的分子生态学初探

第一部分：生物安全信息的建设 生物安全( biosafety)是指经过遗传修饰的生物释放到自然界以后对人类健康和生态环境的影响。随着生物技术的发展，转基因生物的大量涌现，其安全性问题越来越引起人们的关注。本文主要介绍了作者在生物安全信息建设的一些工作，建立了生物安全主页和生物安全文献数据库。对主页和数据库的结构和主要内容进行了详细介绍，生物安全主页介绍了全球转基因生物的发展情况，由此带来的一些潜在风险，国内外对生物安全管理的法规和管理机制，以及对风险评估的技术准则和风险的评价指标体系。生物安全文献数据库收录了600多篇有关的文献。 第二部分：转基因棉花根际土壤微生物分子生态学初探 应用分子生物学方法对转基因棉花和对照非转基因棉花根际土壤微生物的多样性进行了一个生长季的跟踪研究，直接从土壤中提取微生物的总DNA，用PCR方法扩增出原核生物核糖体小亚基16S基因和真核生物ITS片段，并对它们进行了克隆和序列测定，共获得80条16S基因全序列和50条ITS段序列，经过分析它们分别属于7个大类的原核微生物和5个大类的真核生物共计33个属。其中，有44个克隆是传统方法所不能分析的不能被培养的微生物种类。应用邻近法分别绘制了系统关系树状图。 这是首次应用全序列分析的方法研究棉花根际土壤微生物的种类组成。为国际核酸序列数据库贡献130条核酸序列，其中有50条是16S rDNA的全序列。在最后对分子生物学方法在微生物生态学中的应用进行了简短讨论。

Ⅰ濒危植物银杉幼树对生长光强的适应研究、Ⅱ滴灌棉田根区水分对棉花根系生长的调节研究

一． 濒危植物银杉幼树对生长光强的适应研究 银杉（Cathaya argyrophylla）是我国松科中的特有单种属植物，被认为处于濒危状态。在对银杉群落多年调查研究的基础上，针对银杉幼树生长过程对光强的需求特性，开展了银杉幼树对光的适应性研究。试验在人工培育的银杉苗圃地，采用遮荫的方法，设置不同的光环境处理（100%、45%和3%自然光强），利用气体交换技术和叶绿素荧光技术测定了3种光强下银杉叶片光合生理指标的变化，探讨了不同光环境下银杉幼树光合能力在不同季节的变化及对生长光强的响应等。结果表明，在夏季银杉生长旺盛时期，遮荫导致叶片最大光合速率（Pnmax）、羧化效率(CE)明显下降。不同叶龄叶片的下降幅度不同，在3%低光环境下，当年生叶片较1年生叶片降低幅度大。银杉幼树光补偿点(LCP)和光饱和点(L．SP)随生长光强的下降均有所降低，但低光强（3%自然光强）条件下，全晴天时实际的光辐射量高于当年生叶片光补偿点的累积时间约6h，而且距离光饱和的区域相差极大，造成全天碳同化量低，同化物累积少，严重影响了银杉幼树的正常生长：在不同处理中全光强条件下银杉幼树长势最好，45%光强条件下幼树生长减慢。冬季银杉最大光合速率（Pnmax）、羧化效率(CE)值均低于夏季，光补偿点(LCP)和光饱和点(L．SP)也较夏季降低。全光照条件下无论是当年生叶片和1年生叶片，在冬季均出现了轻微光抑制现象，适度遮荫有利于银杉抵御冬季光抑制。无论在遮荫或不遮荫条件下，冬季银杉叶片将所吸收的相对过多光能通过非辐射途经耗散出去，表现出一种光保护策略。 二．滴灌棉田根区水分对棉花根系生长的调节研究 为了探讨膜下滴灌棉花根区土壤水分对棉花根系生长的影响及地上部的关系，揭示膜下滴灌棉花节水高产的生理机理，本试验通过设置不同滴水量处理，控制根区水分供应，研究膜下滴灌棉花根区水分变化对根系生长的调节效应。结果表明，限量滴灌条件下0-40cm土层内相对田间持水率在每次滴水前下降到50%以下，难以满足棉花正常生长对水分的要求;常规滴灌在每次滴水前，根区水分保持在临界水平下限以上，可基本满足棉花根系正常生长的需要，而充分滴灌量处理根区水分偏多。水分对根系生物量的影响表现为，充分滴灌与常规滴灌处理根系生物量差异不显著，两者均显著高于限量滴灌处理。随滴水量的增加，土层中根系分布集中，土壤浅层根量所占比重增加;根系在水平方向上以棉株为中心10cm区域内根量约占总根量的80%以上。根区水分对棉株生理特性也具有一定的调节作用，常规滴灌量的根系活力在生育后期高于充分滴灌量和限量滴灌处理，且深层根系活力所占比例较高。根区水分能够调节棉花根系与地上部生长比例，限量滴灌条件下棉花受到严重的干旱胁迫，虽然根冠比升高，但根系及地上部生物量均明显降低;充分滴灌量条件下单位面积根系总量、总生物学产量在各生育时期均高于常规滴灌量和限量滴灌处理，有较高铃数，但营养器官生物量占总生物量的比例较常规滴灌量处理高，最终实收棉花产量较常规滴灌量处理的低;常规滴灌在各生育时期根冠比适宜，单位重量根系的生产力较高，棉花经济产量高和水分利用效率均较高，增产潜力大。

Recent advances in our knowledge of the biology and conservation of mahseers

Mahseers (Tor khudree), at one time considered to be of single species, are now represented by 6 valid species distributed all over India. Studies on their food habits, ecology of spawning grounds, eggs, larval development and especially methods of artificial propagation have advanced in recent years. Transport of eggs of Tor khudree by air in moist cotton has been possible for easy distribution. Breeding of T. khudree not only by hypophysation, but even without it in small ponds by manipulation of flow of water, exercise and feed has also been possible. Fry and fingerlings of T. khudree are being distributed to many of the states in the country to help conserve the species.

Trends in development in the prawn fishing technique in India: a review

The paper provides some background information about the developments in the prawn fishing technique. The important indigenous gear for prawns are the filtering type nets like the stake nets, boat seines and beach seines with bag. The only indigenous net, which can be compared in design with the modern bottom trawling gear, is the "Thuri Valai" of the Madras Coast. Prawns have also been reported from the catches of gill nets and cast nets. Dredges and beam trawls are simplest among the dragged gear. Even though several earlier attempts were made with the beam trawl, in 1959 season a detailed experiment was made to study the fishing features of this gear. The subsequent experiments conducted were for comparison with otter trawl, towing of 2 beam trawls one behind the other and the use of tickler chain. The initial experiments with otter trawls were mainly directed towards operational aspects. The experiments were to evaluate the effectiveness of fully mechanised and partially mechanised operation, the effect of extra buoyancy of floats and the use of tickler chain. Subsequent developments had been on the structure and design of prawn trawling gear. Effect of long wings and sweeps were tested. Catches of nylon and cotton nets were compared. The sizes of meshes of the cod-end have also been increased.

Experiments on preservation and weathering of net twines

Koura (1963) reporting the results of the comparative studies on different preservation methods of cotton twines stated that "by the difference of rotting in the different waters, not ever one method may be the most economical one". The observations were simultaneously made at Alexandria in Egypt in the sub-tropical region and Cuxhaven in the estuary of the River Elbe in the temperate zone. The course of weathering and effect of immersion in water of man-made fibres have also been mentioned in this communication. Subsequently work on similar lines were extended to Cochin in the tropical region with Cuxhaven and Hamburg as the other places of observation and the results of these studies are presented in this paper.

Growth and survival of Clarias batrachus (Lin.) larvae fed on formulated diets

An experiment was conducted for a period of 28 days using 7-day old C. batrachus larvae of initial total length (±S.D.) of 7.4 ± 0.49 mm and weight (±S.D.) of 2.8 ± 0.75 mg. Five test diets viz. diets I, II, III, IV and V were prepared. Diet I was formulated using 30% fish meal (FM), 20% soybean meal (SM) and 20% Baker's yeast (BY), diet III using 15% FM, 20% SM, 15% cotton seed meal and 30% BY and diet IV using 20% FM, 30% BY and 30% powdered milk. The remaining two diets (diets II and V) were prepared using minced clam meat (96%) and BY (90%) respectively as the sole source of protein. Three replications were used for each treatment. The larvae fed on diet II exhibited significantly (P<0.05) better length gain and specific growth rate (SGR) than those of the larvae fed on diet III and there was no significant difference among the length gain and SGR of the larvae fed on diet I,II,IV and V. The larvae fed on diet II also showed significantly better weight gain and survival than those of the larvae fed on other diets. There was no significant difference between the condition factor of the larvae fed on diet II and IV. However, the larvae fed on diet II showed better condition factor than those of the larvae fed on other diets.

Pathology of mixed infections of saprolegniasis-myxosporidiosis in Indian major carp (Catla catla Ham.)

An outbreak of saprolegniasis in Catla catla in composite carp culture ponds were recorded during winter season. The typical cotton wool growths were observed on whole body surfaces of catla along with sporadic mortality. The fungal invasion was only restricted to skin and no fungal elements were visible in any internal organs after periodic acid schiff staining. On histology, periportal accumulation of mononuclear cells in liver, presence of myxosporidean cysts in antieror kidney, eosinophilic granular cells reaction in submucosa of stomach and intestine, dilated and engorged blood vessels of brain along with sloughing of epidermis and hyperplasia at gill lamellar base were pronounced changes. The possible role of release of Saprolegnia toxin in producing internal organs pathology has been discussed.

棉纤维发育相关基因的克隆和鉴定

棉纤维是棉花子房内的胚珠外珠被上的一种单细胞表皮毛.为了解胚珠表皮细胞发育启动为棉纤维的分子控制机制,我们用同源克隆的方法,从棉纤维发育早期的幼嫩子房和胚珠中克隆到了72个棉花MYB基因.序列分析的结果表明它们属于MYB转录因子家族的55个成员,其中有一个MYB蛋白-GhMYB9的氨基酸序列与控制拟南芥单细胞表皮毛的发育启动基因GL1和WER高度同源.它们之间的序列一致性,不仅表现在保守区-DNA结合区,而且也表现在5’和3’的非保守区内.根据序列相似功能相似的原理,我们推测GhMYB9很可能是控制棉纤维发育启动的一个MYB基因.对GhMYB9的Southern杂交结果表明,该基因在棉花基因组中是单拷贝基因;对它的Northern表达分析可知,该基因在花前5天的子房、花前3天的胚珠、花期及花后3天的胚珠中都有表达,但在花前3天的棉纤维发育启动期（我们的扫描电镜结果）高表达.此外,还构建了一个陆地棉可转化人工染色体TAC文库,并通过PCR的方法从中筛选到了含有GhMYB9的TAC克隆.这些结果为进一步对GhMYB9做功能分析、揭示棉纤维发育启动的分子机制奠定了了事实上的基础.

Fibre optic distributed sensor system for condition monitoring of synthetic ropes

Reliability of the measuring devices is very important problem. Optical fibre sensors are very efficient. The use of optical fibre sensors for monitoring the physical and chemical parameters has been expanding over resent years. These sensors are applied for monitoring the structural integrity of long, parallel lay synthetic ropes. Such ropes are corrosion free, however, their operational lifetime under cyclic load is not well understood and premature failure can occur due to slippage and breakage of yarns within the rope. The monitoring system has been proposed which is based on acoustic detection of yarn breakage. Monitoring the strain and temperature is performed using the array of fibre gratings distributed along the rope length.

Health and disease status of Thai pangas, Pangasius hypophthalmus cultured in rural ponds of Mymensingh, Bangladesh

Thai pangas, Pangasius hypophthalmus is one of the important aquaculture species in Bangladesh. Over the last few years spectacular development has been taking place in Thai pangas farming in Mymensingh district. Due to availability of easy breeding and culture techniques as well as quick return, more and more people are converting their rice fields into pangas farms overnight. The present study was carried out to examine health and disease status of Thai pangas mainly through clinical, histopathological and bacteriological techniques. In addition, for collecting primary data on disease and health status of Thai pangas and the resultant socioeconomic impacts on rural households, questionnaire interview and participatory rural appraisal tools were used with selected farming households in three upazilas of Mymensingh district. The most prevalent diseases as reported by the farmers were red spot, followed by anal protrusion, tail and fin rot, pop eye, dropsy and gill rot. Other conditions like cotton wool type lesion, ulceration and white spot were reported but with lower incidence. Four isolates of Aeromonas hydrophila were recovered from kidney and lesion of diseased fish. Hemorrhage over the body especially near mouth and caudal region was noticed in the fishes associated with aeromonad infection. Internally, kidney, liver and spleen became swollen and enlarged. The isolates varied with their pathogenicity. All the four isolates were sensitive to Nitrofurantoin, Cotrimoxazole and Tetracycline but were resistant to Amoxycilline. An attempt was made to treat diseased fish with extracts from neem leaf, garlic and turmeric. Recovery of infection was monitored through mortality and histopathology. General histopathological changes of different organs were also studied. Extract from neem (Azadirachta indica) leaf gave better result. Telangiectasis, lamellar hypertrophy and hyperplasia hemorrhage, lamellar fusion, necrosis of lamellar epithelial cells, presence of parasites and their cysts were the major pathology of gills. Hemorrhagic lesion, pyknotic nuclei and melanomacrophage centers (MMC) were found in the liver of fish. Major pathologies in kidney of fish included presence of MMC, necrotic and ruptured kidney tubules, severe haemopoietic necrosis, and hemorrhage. The economic loss due to disease in Thai pangas farming was estimated from the difference between expected production and actual production. On an average, Thai pangas farmers of Mymensingh incur a loss of Tk. 23,104/ha/cycle due to fish disease (3.6% of expected total production). The loss, however, varied with location and size of farms, type of farmers and management practices. The study also highlighted fish health management related problems and recommended further work for the development of user-friendly farmer-oriented fish health management packages.

Studies on Raschel knotless netting

Production of knotless webbings is of recent origin in India. An attempt has been made to evaluate the quality of the product consequent to its introduction in the fishing industry. A method has been presented to fix up yarn specification for Raschel knotless nettings equivalent to a given knotted netting.

Observation on the lunar and tidal influence on gill netting in the Bay of Bengal

The effect of lunar and tidal influence on the landing of pelagic fish with drift gill nets has been studied for the first time from Bay of Bengal along the Orissa coast. The catchability of the gear was highest during the first quarter and lowest in the fourth quarter of the moon. The variation in catch rates during the four different quarters was not statistically significant. Contrary to the earlier belief, landing with gill net was not poor during the moonlit nights. The extensive use of bluish grey nylon twine matching with seawater in place of indigenous hemp or cotton twine, has largely eliminated the visibility of gear during the brighter phase of the moon. The rate of exploitation was found to be significantly higher during the nights of neap tide, compared to those of spring tide. A plausible explanation for this phenomenon has been offered.

On the optimum buoyancy-weight relation for a bottom trawl

Comparative fishing operations were carried out with 0.55; 0.75 and 1.00 buoyancy-weight relations of bottom trawl in order to find out the optimum relation. Two nets, 18.26 m. (60') two seam nylon and 16.16 m. (23') two seam cotton trawls, were used for the experiment. The results showed that the total catch per trawling hour with 0.75 B-W relation was 16.5% and 32.08% more than that with 0.55 and 1.00 B-W relation for nylon net used. A similar trend was noticed with cotton trawl also as the catch rate with 0.75 B-W relation was 13.89% and 25.78% more than that with 0.55 and 1.00 B-W relations. However, the analysis of catch composition indicated that the off bottom fishes like lactarius, upenoides sp., synagris sp. etc., were of more percentage with 1.00 B-W relation, near bottom fish like saurida, sciaenids etc., were more with 0.75 B-W relation, while the bottom fishes like soles, prawns, skates and rays etc., were more with 0.55 B-W relation.