The Cheshire Meres: a water quality enigma

This is the report on the Cheshire Meres: a Water Quality Enigma by the Institute of Freshwater Ecology from June 1993. The aim of the proposal is to use a well-tested condition-check on key Cheshire Meres, looking at the present nutrient-state and the seasonal structure and relative species-abundances of the phytoplankton. This information will enable the NRA to make some overall appraisal of the advance or otherwise, in trophic condition and its impact upon the perception, use and management of these nationally important sites. The report contains section on the objectives, strategy, timescale, staff and financial of the proposal, plus an appendix with information about the Windermere Profiler and the CV of the Project Leader.

Efeitos comportamentais e endócrinos do tratamento com a vareniclina em um modelo experimental de exposição à nicotina durante a adolescência

A nicotina é considerada o principal componente psicoativo do tabaco e esta induz seus efeitos farmacológicos centrais atuando em receptores nicotínicos colinérgicos (nAChRs). A maturação dos sistemas colinérgicos é consolidada durante o período da periadolescência, o que sugere que o cérebro do adolescente é vulnerável aos efeitos de estimulantes colinérgicos. Dados os efeitos deletérios do consumo de tabaco, incluindo a dependência, têm sido desenvolvidas estratégias terapêuticas para facilitar a interrupção do uso. A mais recente é o uso da vareniclina, um agonista parcial dos nAChRs α4β2. Pouco se sabe sobre os efeitos da exposição à nicotina na adolescência e menos ainda dos efeitos de curto e longo prazos dos tratamentos disponíveis para a reversão da dependência ainda durante este período. Neste sentido, os objetivos desse trabalho foram o de estudar os comportamentos associados à ansiedade e busca por novos estímulos e a função adrenal em animais expostos à fumaça do cigarro durante a adolescência e subsequentemente tratados com procedimento para reversão de dependência à nicotina. Esse estudo utilizou 150 camundongos Suíços adolescentes (de ambos os sexos). Os animais foram expostos à solução aquosa de nicotina (50g/ml - NIC) ou de sacarina (2% - SAC) v.o. do 30o dia de vida pós-natal (PN) à PN45 e submetidos aos seguintes tratamentos por gavagem de PN45 à PN56: 1) vareniclina (0,1 ou 1,0 mg/kg/dia, VAR1 e VAR2 respectivamente); 2) veículo (VEH); 3) nicotina na dose utilizada entre PN30 e PN45 (NIC). Sete grupos experimentais foram utilizados: SACVEH, SACVAR1, SACVAR2, NICVEH, NICVAR1, NICVAR2 e NICNIC. Em PN55, os animais foram submetidos ao teste do labirinto em cruz elevado (LCE) por 5 min, e duas depois ao teste do campo vazado (CV) também por 5 min. Os tecidos coletados em PN56 (após o sacrifício dos animais) foram: adrenal esquerda para a verificação do conteúdo de catecolaminas, adrenal direita para avaliação da expressão da tirosina hidroxilase e soro para a dosagem de corticosterona. Os nossos resultados demonstraram que no final da vigência do tratamento não foram encontradas diferenças entre os grupos no comportamentos associados à ansiedade do LCE e nos associados à busca por novos estímulos no CV. Por outro lado, a análise do número de orifícios explorados no centro do CV, também utilizado como medida de ansiedade, sugerem que a vareniclina por si só e a exposição continuada à nicotina são ansiogênicas mas que que o tratamento com vareniclina após a exposição à nicotina mantém os resultados dentro da normalidade. Do ponto de vista endócrino, o conteúdo adrenal de catecolaminas foi corrigido pelo tratamento com vareniclina, os níveis séricos de corticosterona foram aumentados pela exposição à nicotina enquanto o tratamento com vareniclina aumentou a expressão de tirosina hidroxilase. Nosso estudo indica que, no final da vigência do tratamento, tanto aspectos comportamentais como endócrinos estão significativamente afetados em nosso modelo de exposição à nicotina durante a adolescência e tratamento subsequente com vareniclina. Estudos futuros deverão avaliar estes parâmetros em períodos posteriores, com o objetivo de verificar se as alterações observadas persistem na vida adulta.

Efeito da estimulação transcraniana por corrente contínua sobre o desempenho físico: análise das respostas cerebrais, metabólicas e perceptivas durante e após exercícios aeróbio e de força

Estimulação transcraniana por corrente contínua (ETCC) sobre áreas corticais pré-selecionadas, tem aumentado o desempenho físico de diferentes populações. Porém, lacunas persistem no tocante aos mecanismos subjacentes à estes efeitos. Assim, a presente tese objetivou: a) investigar os efeitos da ETCC anódica (aETCC) e placebo (Sham) no córtex motor (CM) de indivíduos saudáveis sobre o desempenho de força máxima; b) comparar os efeitos da ETCC sobre a produção de força máxima e estabilidadade da força durante exercícios máximo e submáximo em sujeitos hemiparéticos e saudáveis; c) investigar o efeito da ETCC sobre a conectividade funcional inter-hemisférica (coerência eletroencefalográfica cEEG) do córtex pré-frontal (CPF), desempenho aeróbio e dispêndio energético (EE) durante e após exercício máximo e submáximo. No 1 estudo, 14 adultos saudáveis executaram 2 sessões de exercício máximo de força (EMF) dos músculos flexores e extensores do joelho dominante (3 séries de 10 rep máximas), precedidos por aETCC ou Sham (2mA; 20 mim). aETCC não foi capaz de aumentar o trabalho total e pico de torque (PT), resistência à fadiga ou atividade eletromiográfica durante o EMF. No 2 estudo, 10 hemiparéticos e 9 sujeitos saudáveis receberam aETCC e Sham no CM. O PT e a estabilidade da força (coeficiente de variação - CV) foram avaliados durante protocolo máximo e submáximo de extensão e flexão unilateral do joelho (1 série de 3 reps a 100% do PT e 2 séries de 10 reps a 50% do PT). Nenhuma diferença no PT foi observada nos dois grupos. Diminuições no CV foram obervadas durante a extensão (~25-35%, P<0.001) e flexão de joelho (~22-33%, P<0.001) após a aETCC comparada com Sham nos hemiparéticos, entretanto, somente o CV na extensão de joelhos diminuiu (~13-27%, P<0.001) nos saudáveis, o que sugere que aETCC pode melhorar o CV, mas não o PT em sujeitos hemiparéticos. No 3 estudo, 9 adultos saudáveis realizaram 2 testes incrementais máximos precedidos por aETCC ou Sham sobre o CPF com as respostas cardiorrespiratórias, percepção de esforço (PSE) e cEEG do CPF sendo monitoradas. O VO2 de pico (42.64.2 vs. 38.23.3 mL.kg.min-1; P=0,02), potência total (252.776.5 vs. 23773.3 W; P=0,05) e tempo de exaustão (531.1140 vs. 486.7115.3 seg; P=0,04) foram maiores após aETCC do que a Sham. Nenhuma diferença foi encontrada para FC e PSE em função da carga de trabalho (P>0,05). A cEEG do CPF aumentou após aETCC vs. repouso (0.700.40 vs. 0.380.05; P=0,001), mas não após Sham vs. repouso (0.360.49 vs. 0.330.50; P=0,06), sugerindo que a aETCC pode retardar a fadiga aumentando a conectividade funcional entre os hemisférios do CPF e desempenho aeróbio durante exercício exaustivo. No 4 estudo, o VO2 e EE foram avaliados em 11 adultos saudáveis antes, durante a aETCC ou Sham no CPF e 30 min após exercício aeróbio submáximo isocalórico (~200kcal). Diferenças não foram observadas no VO2 vs. repouso durante aETCC e Sham (P=0.95 e P=0.85). Porém, a associação entre exercício e aETCC aumentou em ~19% o EE após ao menos, 30 min de recuperação após exercício quando comparada a Sham (P<0,05).

三种兰科植物的保护遗传学研究

本研究在野外调查的基础上，采用随机扩增多态DNA (RAPD)分析和形态学方法，研究了我国三种珍稀濒危兰科植物硬叶兜兰（Paphiopedilummicranthum）、麻栗坡兜兰（P. malipoense）和独花兰（Changnienia amoena）的遗传多样性与群体遗传结构，主要结果如下： 1．采用1 2个引物对分布于我国云贵地区的4个硬叶兜兰群体共161个体进行RAPD扩增和分析，得出物种水平的多态条带百分率（PPB）为71.6%，Nci的基因多样度(h)为0.217，Shannon多样性指数（1)为0.3301;4个群体的平均多样性水平为PPB=45.2%，h=0.1457，1= 0.2204：低于远交兰花的平均水平。分子方差分析(AMOVA)表明，在总遗传变异中，群体间遗传变异占20.31%．群体内占79.69%;POPGENE给出的基因分化系数 （Gst）为0.2958;遗传分化略高于远交物种的平均水平。空间自相关分析表明，所检测的两个群体中存在明显的空间结构，基因型在群体中以不同的小斑块存在。遗传距离和空间距离不存在相关关系。 2．用于麻栗坡兜兰的RAPD引物同上，但取样范围只有贵州的2个群体共10个个体。就所研究的个体柬看，麻栗坡兜兰的遗传多样性明显低于远交兰花物种的平均水平。物种水平上，多态条带百分率(PPB)为49.5%。Nei的基因多样度（h）为0. 1174， Shannon多样性指数(I)为0.1764：在群体水平上，上述三个指标的平均值则分别为12. 75%、0.0486和0.0712，均大大低于硬叶兜兰。然而，尽管作了种种努力，麻栗坡兜兰的取样个体数量仍很少，因此所得结果可能会有误差。 3．用16个引物对分布于河南、湖北、湖南、江西4个省11个独花兰群体共216个体进行了RA PD扩增和分析，独花兰在物种水平PPB=80. 7%，h=0.197．1=0. 3116;在群体水平，上述三个指标的平均值则分别为40. 9%、0.1247和0. 1902，均低于远交兰花的平均水平。AMOVA分析表明，11个独花兰群体间的遗传变异占43.48%，群体内的占56.52%：在神农架和新宁地区内部，群体间的遗传变异分别占13.68%和49.3g%(AMOVA)。POPGENE给出的11个群体的基因分化系数(Gst)为0.3580．神农架和新宁地区内的Gst，值分别为0.1194和0.2597。可见，群体间的遗传分化明显高于远交物种的平均水平。空间自相关分析表明，独花兰的遗传变异在群体内不存在明显的空间结构。群体之间的遗传距离和空间距离不存在相关关系。 4．对独花兰7个群体形态性状的分析发现，12个形态性状在群体内均有较高的变异性，cv值变动于0.022-0.30O。庐山群体(LS)在所有性状上的平均值均为最高。营养性状和花部性状的变异性基本一致。除花葶长和花距直径与某些花部性状之间没有显著的相关关系外，各性状之间均有显著的相关性。对XN4群体的统计没有发现假磷茎数目与其他性状之间存在显著相关性。 根据以上对硬叶兜兰、麻粟坡兜兰和独花兰遗传多样性和群体遗传结构韵研究，结合其他方面的资料;对三种兰花的濒危机制进行了初步的分析。首先，人为采挖和破坏是导致这些兰花物种濒危的直接原因，尤其是麻栗坡兜兰。其次， 适宜兰花生存的生境正在只益萎缩、退化和片段化。这两方面因素的共同作用导致上述兰花群体的数目和规模日益下降，由此引发的遗传多样性降低和遗传结构的改变进一步加剧其濒危状况。对于独花兰而言，较低的繁殖能力又使其生存状态雪上加霜。针对三个物种不同的繁殖特性和遗传学状况，提出如下保护措施。(1)硬叶兜兰由于繁殖能力较强、现存个体尚多，遗传多样性损失不甚严重，因此以保护其所在的生境为基础、实施原位保护，是比较合适的保护策略。(2)麻粟坡兜兰目前受破坏程度非常严重;所剩个体很少，遗传多样性较低，已经很难进行有效的原位保护。因此;应利用迁地保护手段抢救目前尚存的个体。(3)独花兰的繁殖能力较弱，因此在保护生境和严禁采摘的基础上，可采用人工授粉等方式，提高结实率、增加繁殖效率，促使其复壮：在进行迁地保护时，则应注意不同群体间存在较大遗传变异而群体内多样性较低这一现实。

小麦肉桂酰辅酶A还原酶（CCR）基因的分离和功能分析

作为植物界广泛存在的一类酚类聚合物，木质素是陆生植物正常生长发育过程中非常重要的生物大分子，而且与人类的生活息息相关。利用分子生物学手段和基因工程方法，从小麦中分离木质素生物合成途径的关键酶-肉桂酰辅酶A还原酶基因（CCR），研究肉桂酰辅酶A还原酶基因在木质素代谢途径中的调控规律，从其催化的限速步骤入手，来调控木质素的合成，有效的改变木质素的组成、含量和结构，是改善木质素在植物生长发育中的作用乃至开发木质素资源的关键所在。本文就小麦肉桂酰辅酶A还原酶基因的分离、表达特征及其在木质素合成途径中的作用开展了研究工作。 首先用RACE方法从小麦中克隆了CCR的两个cDNA的部分序列，序列分析表明它们编码的蛋白具有CCR的典型特点，GC含量高于均60%，两者在核酸水平和蛋白水平的同源性为76%和 69%，证明在小麦中至少存在着两个CCR基因。通过 RT-PCR和Northern 杂交确定W-cr6和W-cr19在小麦的发育中具有不同的表达特征，W-cr6主要在茎中表达，而W-cr19的表达集中在根中。以W-cr6为探针，从cDNA文库中筛选到一个全长1317bp的cDNA，命名为TaCCR1。TaCCR1包括开放阅读框 (ORF) 1047bp、5′端侧翼 72bp和3′端侧翼198bp的非翻译序列。TaCCR1能够编码由349个氨基酸组成的蛋白质，预期的分子量为37.4kD。同源性比较显示TaCCR1基因在核酸水平和蛋白质水平与其他物种的CCR基因的同源性高于60%。 为了分析CCR在木质素合成中的作用，用TaCCR1构建了用于转化烟草的正义和反义表达载体pStCCR和pAtCCR、用于转化小麦的正义和反义表达载体pBSC1和pBAC1。通过农杆菌介导得到了30株反义转基因烟草和12株正义转基因烟草。由于外源基因的抑制作用，转基因烟草在形态、木质素组成和含量、木质部显微结构上都程度不同的发生了变化。正义和反义的转基因株系呈现出株型矮化、木质素含量下降、木质部导管细胞壁受到破坏等现象。同时利用花粉管通道法转化小麦种子5000多粒，部分处理经过初步的PCR和 Southern分子鉴定获得了1株转基因株系，需要对其遗传、生理和形态特征做进一步的研究。 本文还对木质素对小麦茎杆的机械强度的影响做了初步的探讨，得到的结果是小麦茎杆的木质素含量、维管束的数量、茎杆有效的横界面积与其最大弯曲应力存在着正相关，而维管束的结构、密度对茎杆的最大弯曲应力没有明显的影响，从而为通过CCR基因来改善小麦茎杆的抗倒特性建立了生理学基础。

牵牛光周期诱导过程中特异蛋白质的出现

日本紫花牵牛（pharbitis nil CV violet）幼苗经三种不同光周期处理后（I. 16小时暗处理;II. 16小时持续光照;III、16小时暗处理中间施加10分钟光间断）不同时间取子叶提取蛋白质，双向电泳分析后发现，暗处理后24小时子叶中出现一特异蛋白质（MW：19KD，pI4.5）。暗处理后48小时仍能观察到此特异蛋白质。 通过观察光周期诱导前后不同时期去子叶或向子叶施加抑制剂对牵牛开花的抑制作用，推测与光周期诱导相关的mRNA是在暗处理期间合成的，而特异蛋白质的合成则持续到暗期结束后的24小时。在牵牛子叶内进行的光周期诱导需40小时才得以完成（16小时暗处理+随后的24小时）。 通过研究经三种不同光周期处理的牵牛子叶中线粒体在离体情况下蛋白质的合成，观察到经暗处理的牵牛子叶中，线粒体在体外多合成了三种蛋白质（与经NB处理和CL处理的植株相比）。从此推测牵牛经暗处理后，子叶中特异蛋白质的产生有特异时空性。

冬小麦春化过程中低温诱导的开花特异mRNA及蛋白质的合成

本实验以冬小麦“农大139”（T. aestivum L. cv. Nongda 139）为材料对春化作用诱导开花的分子机制作了一些探讨，主要结果如下： 1. 应用SDS-PAGE及高分辨双向电泳技术，比较了冬小麦给予春化、脱春化及超期春化处理后的电泳图谱，并结合对春小麦对照样品的分析，结果发现有一些蛋白质和春化作用紧密相关，它们随春化而出现、随脱春化而消失，并在不经低温处理即可以开花的春小麦对照中存在。也就是说，这些开花特异的蛋白质（FSPs）的存在或在诱导下的合成和小麦抽穗开花能力的获得存在一种正相关，因此推测它们在冬小麦由营养生长状态向生殖生长状态转变的过程中起了关键性的作用。 2. 从不同处理的及对照样品中提取mRNA进行体外翻译的结果表明：春化作用过程中低温诱导了mRNA组分的变化，其中一些新产生的mRNA种类与春化诱导的开花能力的获得呈高度相关，即它们是开花特异的（Flower Specific），它们中有的只在春化的特定时期存在并起作用。 3．比较体内分析及体外翻译的结果发现，一些开花特异蛋白质（FSPs）可以同时在体内提取物及体外翻译产物中检测到，因此，春化作用中开花特异蛋白质诱导合成的调节很可能发生在转录水平上。 4．基于以上结果的分析可以推测春化诱导开花是低温导致了开花特异基因表达的结果，超期春化的效应不能被脱春化所逆转则系编码这些开花特异蛋白质的基因在长期低温条件下转变成了组成性表达所致。 有关低温诱导产生的开花特异蛋白质的性质与功能及编码这些蛋白质的基因尚需进一步研究。

长日照处理对短日植物紫花牵牛开花的仰制

本文以日本紫花牵牛花为材料，观察分析了其对不同光周期的反应应特性以及不同光周期处理条件下子叶蛋白质和mRNA的变状况。实验结果归纳如下：1、经不同天数短日照处理的幼苗导致植株开花特性明显不同。2、实验结果表明，12小时暗期长度可能是诱导光周期的临界暗期长度。3、经双向电泳分析观察到，长日照处理的牵牛子叶内存在着分子量分别为16.5KD(PI4.1), 16.5KD(PI4.2), 21.6KD(PI8.8)及21.7KD(PI8.3)四种蛋白质，它们在短日诱导条件下消失，表明这些蛋白质的存在可能与抑制花芽分化有关。4、提取经短日照诱导和连续光照子叶内的PolY(A~+)RNA, 进行体外翻译，观察表明光周期诱导过程是在转录水平上的调节。短日照处理不仅有促进芽分化的作用，同时还有消除长日照的抑制效应。

普通烟草与毛曼陀罗族间体细胞杂交的研究

采用PEG／DMSO融合法，从普通烟草(Nicotiana tabacum L.cv.Xanthi nc)叶肉和毛曼陀罗(Datura innoxia Mill.)茎或叶愈伤组织原生质体融合获得I5株花盆中健康生长的族间体细胞杂种植株;其中11株是在没有选择压力的条件下获得，4株则是在有IOA和R6G选择系统存在下获得。同时获得一株试管开花且形态异常的植株和一株花盆中生长的嵌合植株。lOmM IOA和I5μ g／ml R6G均能分别有效地抑制烟草叶肉和毛曼陀罗愈伤组织原生质体的分裂;10% DMSO能显著提高原生质体融合率;PEG种类并不重要，但浓度则很重要;BAP较ZT，KT对植株分化有更好的诱导效果。杂种的形态、细胞、同工酶、Southern杂交，花粉育性分析结果如下：1、l5株杂种较双亲普遍株型矮小，生长缓慢，形态接近烟草但不很正常，根据形态特征可分为两种类型：(1)共有8株，其叶片大小、形状、颜色、开花习惯、花类型（单花）等均与毛曼陀罗接近，但子房败育;(2)共有7株，其株型、叶片形状、颜色、光滑度、花形状、类型（圆锥状花序）、颜色更接近烟草，但少数杂种开单花或先单花后圆锥状花序或先单花后两种花并存，且开花时间不一，部分子房败育。2、杂种染色体数目大都在60～90之间，个别者较少(48条)或较多(125条)，没有一株为双二倍体(2n=96)，并全部为混倍体。3、15株杂种植株均有双亲的细胞色素氧化酶同工酶特征谱带;大部分都有双亲的过氧化物酶同工酶特征谱带，少都仅具烟草的谱带。4、Hae Ⅲ／水稻rDNA的Southern杂交分析表明杂种1较双亲多一条谱带，杂种2较双亲也多一条弱带，其它杂种尚待定。5、花粉活力测定表明毛曼陀罗（种子再生而来）的为99%，烟草（原生质体再生而来）为80～90%，而杂种的为24～61%，育性普遍低于双亲。

运用减法杂交、差异筛选与信使RNA差别显示聚合酶链式反应克隆冬小麦春化相关基因

对于某些一年生或二年生高等植物，春化作用是诱导其成花的一个重要的环境因子。冬小麦春化进程中存在着一个核酸代谢的关键期，利用分子生物学技术分离特异表达的基因是研究春化诱导成花机理的一个突破口。 利用TRIzol试剂快速提取冬小麦燕大1817(Triticum aestivum L. cv Yanda 1817)未春化、春化4d、春化20d、5d脱春化的胚芽中的总RNA，去除污染的DNA后，将引物P_1(5'TTTTTTTTTTTCA3')、P_2(5'TTTTTTTTTTCC3')与10个碱基的随机引物OPF_1-OPF_(20)、OPG_1-OPG_(20)组成80个引物对，对不同来源的RNA进行差别显示，共显示了大约10,000种mRNA，结果发现了两个仅在春化20d这一关键期表达而在未春化、春化4d、5d脱春化时不表达的春化相关基因(VRG)VRG49与VRG54。Northern分析进一步表明这两个基因仅与春化20d的冬小麦RNA有杂交信号。将VRG49与VRG54亚克隆于pGEM-4Z载体上，利用T_7测序系统获得了VRG49和VRG54的DNA序列，它们的长度分别为307bp与169bp。 春化21d的冬小麦京冬1号(T. aestivum L. cv Jingdong No. 1)胚芽的mRNA在逆转酶作用下反转录成sscDNA杂交，将过量的未春化、脱春化的mRNA与sscDNA杂交，运用磁珠法分离出未杂交上的sscDNA，以特异的sscDNA为模板，用DNA聚合酷I合成了dscDNA。通过对dscDNA内部EcoRI位点的甲基化、末端补平、EcoRI接头的安装、连接进入λgt10载体的EcoRI位置，以及运用包装系统进行体外包装，建立了库容为4 * 10~6pfu的富集低温诱导的冬小麦cDNA噬菌体文库。用来源于未春化、春化21d、脱春化的冬小麦mRNA合成3种cDNA探针，对噬菌斑进行原位杂交，结果筛选出了3个春化相关基因(VRG)VRG79、VRG111和VRG231。Dot blotting与Northern分析表明VRG79仅在冬小麦春化关键期21d表达。运用PCR方法从λgt10DNA中扩增出VRG79片断并亚克隆于PUC18载体上，通过T_7测序系统获得了VGR79的序列，其包括349个碱基。 通过Internet将VRG49、VRG54、VRG79与GenBank、EMBL、DDBJ、PBD中的序列进行同源性分析，结果发现这些基因至少是在植物中新发现的基因，对这些基因推测的一些功能也进行了讨论。

转基因植物生产生物可降解塑料PHB的研究-phbA功能鉴定、多价表达载体的构建及转基因植物的获得

聚-β-羟基链烷酸（PHA）是许多微生物作为碳源、能源的一类贮藏性聚酯，具有广泛的应用价值。该聚酯可被微生物完全降解且有与塑料相似的性质，因而研究并提高PHA在植物中的合成为解决环境污染提供了新的解决途径。 聚-β-羟基于酸酯（PHB）是研究的最早、研究的最清楚的一种PHA。用聚合酶链式反应扩增并克隆了真养产碱杆菌（Alcaligenes eutrophus）中合成PHB的一个关键酶——3-酮硫裂解酶基因phbA。DNA序列分析表明所克隆的基因与国外报道序列同源性很高，只有一个碱基对的区别。为了检测该基因的功能及导肽的定位效率，构建了带有导肽基因的组成型表达载体，由根癌农杆菌介导转化烟草（Nicotiana tabacum cv. Wisconsin 38）得到转基因植株。蛋白质电泳结果表明导肽可以将外源蛋白定位于质体，phbA基因能翻译成相应大小的蛋白。酶活性分析证实了转基因烟草中phbA编码的3-酮硫裂解酶可以催化乙酰-CoA合成乙酰乙酰-CoA。 将携有导肽序列的phbC（编码PHB合酶）和phbB（编码乙酰乙酰-CoA还原酶）连入pBIB-HYG得到组成型表达载体pZCB，用冻融法转入根癌农杆菌，介导转化烟草。烟草为已获得的具有卡那霉素抗性整合并表达phbA的转基因烟草。通过二次转化将携有潮霉素抗性的phbB基因和phbC基因导入已整合phbA的烟草，各基因均由质体导肽控制，最后得到整合PHB合成的三个酶基因的转基因烟草。转基因烟草经PCR、PCR-Southern检测，初步确定整合phbB和phbC烟草植株。以气相色谱初步分析，转基因烟草中PHB的含量可达鲜重的0.233%。 结果表明phbB和phbC基因可以在真核表达系统中编码相应的蛋白。通过色素分析、荧光动力学等手段分析了PHB在叶绿体中的累积对其功能的影响。 为了提高底物乙酰-CoA的供应能力及减少惰性聚酯对植物体的伤害，分离了种子特异性启动子和质体导肽序列，利用忆经克隆的合成PHB的三个关键酶基因，通过一系列DNA重组，分别构建了含有种子特异性启动子的嵌合phbC、phbB的二价表达载体pSCB及嵌合phbC、phbA、phbB的三价表达载体pSCAB，并由导肽将基因表达产物定位于质体。经根癌农杆菌介导转化油菜（Brassica napus L.) H165，获得转基因油菜植株，并进行了PCR、Southern blot及RT-PCR-DNA杂交等分检测。结果表明，三基因已经分别整合到相应的转基因油菜中，并已在转录水平表达。同时转化了油菜不育系、恢复系和保持系，获得批量转化株，并移入温室栽培。

Small-boat surveys for coastal dolphins: line-transect surveys of Hector’s dolphins (Cephalorhynchus hectori)

Management of coastal species of small cetaceans is often impeded by a lack of robust estimates of their abundance. In the Austral summers of 1997−98, 1998−99, and 1999−2000 we conducted line-transect surveys of Hector’s dolphin (Cephalorhynchus hectori) abundance off the north, east, and south coasts of the South Island of New Zealand. Survey methods were modified for the use of a 15-m sailing catamaran, which was equipped with a collapsible sighting platform giving observers an eye-height of 6 m. Eighty-six percent of 2061 km of survey effort was allocated to inshore waters (4 nautical miles [nmi] or 7.4 km from shore), and the remainder to offshore waters (4−10 nmi or 7.4–18.5 km from shore). Transects were placed at 45° to the shore and spaced apart by 1, 2, 4, or 8 nmi according to pre-existing data on dolphin density. Survey effort within strata was uniform. Detection functions for sheltered waters and open coasts were fitted separately for each survey. The effect of attraction of dolphins to the survey vessel and the fraction of dolphins missed on the trackline were assessed with simultaneous boat and helicopter surveys in January 1999. Hector’s dolphin abundance in the coastal zone to 4 nmi offshore was calculated at 1880 individuals (CV=15.7%, log-normal 95% CI=1384−2554). These surveys are the first line-transect surveys for cetaceans in New Zealand’s coastal waters.

Distribution and abundance of humpback whales (Megaptera novaeangliae) and other marine mammals off the northern Washington coast

We examined the summer distribution of marine mammals off the northern Washington coast based on six ship transect surveys conducted between 1995 and 2002, primarily from the NOAA ship McArthur. Additionally, small boat surveys were conducted in the same region between 1989 and 2002 to gather photographic identification data on humpback whales (Megaptera novaeangliae) and killer whales (Orcinus orca) to examine movements and population structure. In the six years of ship survey effort, 706 sightings of 15 marine mammal species were made. Humpback whales were the most common large cetacean species and were seen every year and a total of 232 sightings of 402 animals were recorded during ship surveys. Highest numbers were observed in 2002, when there were 79 sightings of 139 whales. Line-transect estimates for humpback whales indicated that about 100 humpback whales inhabited these waters each year between 1995 and 2000; in 2002, however, the estimate was 562 (CV= 0.21) whales. A total of 191 unique individuals were identified photographically and mark recapture estimates also indicated that the number of animals increased from under 100 to over 200 from 1995 to 2002. There was only limited interchange of humpback whales between this area and feeding areas off Oregon and California. Killer whales were also seen on every ship survey and represented all known ecotypes of the Pacific Northwest, including southern and northern residents, transients, and offshore-type killer whales. Dall’s porpoise (Phocoenoides dalli) were the most frequently sighted small cetacean; abundance was estimated at 181−291 individuals, except for 2002 when we observed dramatically higher numbers (876, CV= 0.30). Northern fur seals (Callorhinus ursinus) and elephant seals (Mirounga angustirostris) were the most common pinnipeds observed. There were clear habitat differences related to distance offshore and water depth for different species.

Magnitude and distribution of sea turtle bycatch in the sea scallop (Placopecten magellanicus) dredge fishery in two areas of the northwestern Atlantic Ocean, 2001–2002

In May 2001, the National Marine Fisheries Service (NMFS) opened two areas in the northwestern Atlantic Ocean that had been previously closed to the U.S. sea scallop (Placopecten magellanicus) dredge fishery. Upon reopening these areas, termed the “Hudson Canyon Controlled Access Area” and the “Virginia Beach Controlled Access Area,” NMFS observers found that marine turtles were being caught incidentally in scallop dredges. This study uses the generalized linear model and the generalized additive model fitting techniques to identify environmental factors and gear characteristics that influence bycatch rates, and to predict total bycatch in these two areas during May-December 2001 and 2002 by incorporating environmental factors into the models. Significant factors affecting sea turtle bycatch were season, time-of-day, sea surface temperature, and depth zone. In estimating total bycatch, rates were stratified according to a combination of all these factors except time-of-day which was not available in fishing logbooks. Highest bycatch rates occurred during the summer season, in temperatures greater than 19°C, and in water depths from 49 to 57 m. Total estimated bycatch of sea turtles during May–December in 2001 and 2002 in both areas combined was 169 animals (CV=55.3), of which 164 (97%) animals were caught in the Hudson Canyon area. From these findings, it may be possible to predict hot spots for sea turtle bycatch in future years in the controlled access areas.

Quantifying annual variation in catchability for commercial and research fishing

Biomass indices, from commercial catch per unit of effort (CPUE) or random trawl surveys, are commonly used in fisheries stock assessments. Uncertainty in such indices, often ex-pressed as a coefficient of variation (CV), has two components: observation error, and annual variation in catchability. Only the former can be estimated directly. As a result, the CVs used for these indices either ignore the annual-variation component or assume a value for it (often implicitly). Two types of data for New Zealand stocks were examined: 48 sets of residuals and catchability estimates from stock assessments using either CPUE or trawl survey indices; and biomass estimates from 17 time series of trawl surveys with between 4 and 25 species per time series. These data show clear evidence of significant annual variation in catchability. With the trawl survey data, catchability was detectably extreme for many species in about one year in six. The assessment data suggest that this annual variability typically has a CV of about 0.2. For commercial CPUE the variability is slightly less, and a typical total CV (including both components) of 0.15 to 0.2. This is much less than the values of 0.3 to 0.35 that have commonly been assumed in New Zealand. Some estimates of catchability are shown to be implausible.