Anticancer property of Bryophyllum pinnata (Lam.) Oken. leaf on human cervical cancer cells

Background: Bryophyllum pinnata (B. pinnata) is a common medicinal plant used in traditional medicine of India and of other countries for curing various infections, bowel diseases, healing wounds and other ailments. However, its anticancer properties are poorly defined. In view of broad spectrum therapeutic potential of B. pinnata we designed a study to examine anti-cancer and anti-Human Papillomavirus (HPV) activities in its leaf extracts and tried to isolate its active principle. Methods: A chloroform extract derived from a bulk of botanically well-characterized pulverized B. pinnata leaves was separated using column chromatography with step-gradient of petroleum ether and ethyl acetate. Fractions were characterized for phyto-chemical compounds by TLC, HPTLC and NMR and Biological activity of the fractions were examined by MTT-based cell viability assay, Electrophoretic Mobility Shift Assay, Northern blotting and assay of apoptosis related proteins by immunoblotting in human cervical cancer cells. Results: Results showed presence of growth inhibitory activity in the crude leaf extracts with IC50 at 552 mu g/ml which resolved to fraction F4 (Petroleum Ether: Ethyl Acetate:: 50: 50) and showed IC50 at 91 mu g/ml. Investigations of anti-viral activity of the extract and its fraction revealed a specific anti-HPV activity on cervical cancer cells as evidenced by downregulation of constitutively active AP1 specific DNA binding activity and suppression of oncogenic c-Fos and c-Jun expression which was accompanied by inhibition of HPV18 transcription. In addition to inhibiting growth, fraction F4 strongly induced apoptosis as evidenced by an increased expression of the pro-apoptotic protein Bax, suppression of the anti-apoptotic molecules Bcl-2, and activation of caspase-3 and cleavage of PARP-1. Phytochemical analysis of fraction F4 by HPTLC and NMR indicated presence of activity that resembled Bryophyllin A. Conclusions: Our study therefore demonstrates presence of anticancer and anti-HPV an activity in B. pinnata leaves that can be further exploited as a potential anticancer, anti-HPV therapeutic for treatment of HPV infection and cervical cancer.

Lipopeptides from the Banyan Endophyte, Bacillus subtilis K1: Mass Spectrometric Characterization of a Library of Fengycins

Mass spectrometric analysis of a banyan endophyte, Bacillus subtilis K1, extract showing broad spectrum antifungal activity revealed a complex mixture of lipopeptides, iturins, surfactins, and fengycins. Fractionation by reversed-phase high performance liquid chromatography (HPLC) facilitated a detailed analysis of fengycin microheterogeneity. Matrix assisted laser desorption ionization (MALDI) and electrospray ionization (ESI) mass spectrometric studies permitted the identification of several new fengycin variants. Four major sites of heterogeneity are identified: (1) N-terminus beta-hydroxy fatty acid moiety, where chain length variation and the presence of unsaturation occur, (2) position 6 (Ala/Val/Ile/Leu), (3) position 10 (Val/Ile) within the macrocyclic ring, and (4) Gln to Glu replacement at position 8, resulting in fengycin variants that differ in mass by 1 Da. Diagnostic fragment ions provide a quick method for localizing the sites of variation in the macrocycle or the linear segment. Subsequent establishment of the sequences is achieved by MS/MS analysis of linear fengycin species produced by hydrolysis of the macrocyclic lactone. Unsaturation in the fatty acid chain and the presence of linear precursors in the B. subtilis K1 extract are also established by mass spectrometry. The anomalous distribution of intensities within isotopic multiplets is a diagnostic for Gln/Glu replacements. High resolution mass spectrometry facilitates the identification of fengycin species differing by 1 Da by localizing the variable position (Gln(8)/Glu(8)) in the fengycin variants.

Characterisation and bioactivity of oosporein produced by endophytic fungus Cochliobolus kusanoi isolated from Nerium oleander L.

Bioactive compounds comprising secondary metabolites produced by endophytic fungi have wide applications in pharmacology and agriculture. Isolation, characterisation and evaluation of biological activities of secondary metabolites were carried out from Cochliobolus kusanoi an endophytic fungus of Nerium oleander L. The fungus was identified based on 18S rDNA sequence analysis. There are no reports available on the compounds of C. kusanoi hence, antimicrobial metabolite produced by this fungus was extracted and purified by fractionation using hexane, diethyl ether, dichloromethane, ethyl acetate and methanol. Out of all the solvent fractions, the methanol fraction exhibited better antimicrobial activity which was further purified and characterised as oosporein. Oosporein from C. kusanoi exhibited broad spectrum in vitro antimicrobial, antioxidant and cytotoxic activities. The characterisation and antioxidant activity of oosporein from C. kusanoi are reported for the first time.

Medieval pulse of great earthquakes in the central Himalaya: Viewing past activities on the frontal thrust

The Himalaya has experienced three great earthquakes during the last century1934 Nepal-Bihar, 1950 Upper Assam, and arguably the 1905 Kangra. Focus here is on the central Himalayan segment between the 1905 and the 1934 ruptures, where previous studies have identified a great earthquake between thirteenth and sixteenth centuries. Historical data suggest damaging earthquakes in A.D. 1255, 1344, 1505, 1803, and 1833, although their sources and magnitudes remain debated. We present new evidence for a great earthquake from a trench across the base of a 13m high scarp near Ramnagar at the Himalayan Frontal Thrust. The section exposed four south verging fault strands and a backthrust offsetting a broad spectrum of lithounits, including colluvial deposits. Age data suggest that the last great earthquake in the central Himalaya most likely occurred between A.D. 1259 and 1433. While evidence for this rupture is unmistakable, the stratigraphic clues imply an earlier event, which can most tentatively be placed between A.D. 1050 and 1250. The postulated existence of this earlier event, however, requires further validation. If the two-earthquake scenario is realistic, then the successive ruptures may have occurred in close intervals and were sourced on adjacent segments that overlapped at the trench site. Rupture(s) identified in the trench closely correlate with two damaging earthquakes of 1255 and 1344 reported from Nepal. The present study suggests that the frontal thrust in central Himalaya may have remained seismically inactive during the last similar to 700years. Considering this long elapsed time, a great earthquake may be due in the region.

Evaluation of Several Commercial Algicides for Control of Odor-producing Cyanobacteria

The production of certain odorous metabolites is an undesirable attribute of cyanobacteria (blue-green algae) growth in aquaculture ponds [e.g., channel catfish(Ictalurus punctatus)] and in drinking water reservoirs. The most common odorous compounds encountered in catfish aquaculture are geosmin (trans-1,10-dimethyltrans-9-decalol) and 2-methylisoborneol(exo-1,2,7,7-tetramethylbicyclo[2.2.1]heptan-2-ol). These compounds are also frequently encountered worldwide in reservoirs and aqueducts used for municipal drinking water systems(Schrader et al. 2002). In this study, several algicides were evaluated using a rapid bioassay to determine their effectiveness in controlling the MIB-producing cyanobacterium Oscillatoria perornata from a west Mississippi catfish pond and the MIBproducing Pseudanabaena sp. (strain LW397) from Lake Whitehurst, Virginia, used as a city water supply reservoir. The cyanobacterium Oscillatoria agardhii , not a MIB-producer, and the green alga Selenastrum capricornutum , found in catfish ponds in the southeastern United States, were included in the bioassay to help determine potential broad-spectrum toxicity of the commercial products. (PDF has 3 pages.)

Final report of the Support to Regional Aquatic Resources Management (STREAM): a NACA networking initiative

The governing council of Naca has resolved to effect a shift in emphasis from aquaculture development to aquaculture for development. This will require engaging partners from a broad spectrum of government and development agencies, the nature of the information that will need to be gathered and the strategies used for disseminating information and initiating action. The vehicle for operationalising this shift is STREAM - Support to Regional Aquatic Resources Management. This report outlines the nature of the STREAM network, its relationship to NACA's vision, mission, objectives and operating principles, and how STREAM differs from previous NACA's networks. Because STREAM is different, a theoretical basis for network communication is presented along with an outline of the preliminary steps in getting the network up and running. (Pdf contains 33 pages).

Somatisation in primary care: experiences of primary care physicians involved in a training program and in a randomised controlled trial

Background: A new intervention aimed at managing patients with medically unexplained symptoms (MUS) based on a specific set of communication techniques was developed, and tested in a cluster randomised clinical trial. Due to the modest results obtained and in order to improve our intervention we need to know the GPs' attitudes towards patients with MUS, their experience, expectations and the utility of the communication techniques we proposed and the feasibility of implementing them. Physicians who took part in 2 different training programs and in a randomised controlled trial (RCT) for patients with MUS were questioned to ascertain the reasons for the doctors' participation in the trial and the attitudes, experiences and expectations of GPs about the intervention. Methods: A qualitative study based on four focus groups with GPs who took part in a RCT. A content analysis was carried out. Results: Following the RCT patients are perceived as true suffering persons, and the relationship with them has improved in GPs of both groups. GPs mostly valued the fact that it is highly structured, that it made possible a more comfortable relationship and that it could be applied to a broad spectrum of patients with psychosocial problems. Nevertheless, all participants consider that change in patients is necessary; GPs in the intervention group remarked that that is extremely difficult to achieve. Conclusion: GPs positively evaluate the communication techniques and the interventions that help in understanding patient suffering, and express the enormous difficulties in handling change in patients. These findings provide information on the direction in which efforts for improving intervention should be directed.

Resource management of dynamic coastal environments using synoptic measures from remote sensing

In addition to providing vital ecological services, coastal areas of North Carolina provide prized areas for habitation, recreation, and commercial fisheries. However, from a management perspective, the coasts of North Carolina are highly variable and complex. In-water constituents such as nutrients, suspended sediments, and chlorophyll a concentration can vary significantly over a broad spectrum of time and space scales. Rapid growth and land-use change continue to exert pressure on coastal lands. Coastal environments are also very vulnerable to short-term (e.g., hurricanes) and long-term (e.g., sea-level rise) natural changes that can result in significant loss of life, economic loss, or changes in coastal ecosystem functioning. Hence, the dynamic nature, effects of human-induced change over time, and vulnerability of coastal areas make it difficult to effectively monitor and manage these important state and national resources using traditional data collection technologies such as discrete monitoring stations and field surveys. In general, these approaches provide only a sparse network of data over limited time and space scales and generally are expensive and labor-intensive. Products derived from spectral images obtained by remote sensing instruments provide a unique vantage point from which to examine the dynamic nature of coastal environments. A primary advantage of remote sensing is that the altitude of observation provides a large-scale synoptic view relative to traditional field measurements. Equally important, the use of remote sensing for a broad range of research and environmental applications is now common due to major advances in data availability, data transfer, and computer technologies. To facilitate the widespread use of remote sensing products in North Carolina, the UNC Coastal Studies Institute (UNC-CSI) is developing the capability to acquire, process, and analyze remotely sensed data from several remote sensing instruments. In particular, UNC-CSI is developing regional remote sensing algorithms to examine the mobilization, transport, transformation, and fate of materials between coupled terrestrial and coastal ocean systems. To illustrate this work, we present the basic principles of remote sensing of coastal waters in the context of deriving information that supports efficient and effective management of coastal resources. (PDF contains 4 pages)

Single sub-100 attosecond pulse generation in a two-colour time-gating laser field

We theoretically propose a method of generating a single sub-100 attosecond (as) pulse with a two-colour time-gating laser field. The field is synthesized by an 8 fs/800 nm (three optical cycles) pulse and a 24 fs/2400 nm (three optical cycles) pulse with an optimal time delay between them. In our simulation, we obtain a supercontinuum with an extremely broad spectrum of 150 eV and generate an isolated attosecond pulse with 96 as pulse duration without any dispersion compensation.

Engineering enzyme systems by recombination

Homologous recombination is a source of diversity in both natural and directed evolution. Standing genetic variation that has passed the test of natural selection is combined in new ways, generating functional and sometimes unexpected changes. In this work we evaluate the utility of homologous recombination as a protein engineering tool, both in comparison with and combined with other protein engineering techniques, and apply it to an industrially important enzyme: Hypocrea jecorina Cel5a.

Chapter 1 reviews work over the last five years on protein engineering by recombination. Chapter 2 describes the recombination of Hypocrea jecorina Cel5a endoglucanase with homologous enzymes in order to improve its activity at high temperatures. A chimeric Cel5a that is 10.1 °C more stable than wild-type and hydrolyzes 25% more cellulose at elevated temperatures is reported. Chapter 3 describes an investigation into the synergy of thermostable cellulases that have been engineered by recombination and other methods. An engineered endoglucanase and two engineered cellobiohydrolases synergistically hydrolyzed cellulose at high temperatures, releasing over 200% more reducing sugars over 60 h at their optimal mixture relative to the best mixture of wild-type enzymes. These results provide a framework for engineering cellulolytic enzyme mixtures for the industrial conditions of high temperatures and long incubation times.

In addition to this work on recombination, we explored three other problems in protein engineering. Chapter 4 describes an investigation into replacing enzymes with complex cofactors with simple cofactors, using an E. coli enolase as a model system. Chapter 5 describes engineering broad-spectrum aldehyde resistance in Saccharomyces cerevisiae by evolving an alcohol dehydrogenase simultaneously for activity and promiscuity. Chapter 6 describes an attempt to engineer gene-targeted hypermutagenesis into E. coli to facilitate continuous in vivo selection systems.

Interpretações de linguagens artísticas em Gerd Bornheim

Este estudo propõe uma leitura histórico-cultural das interpretações de Gerd Bornheim (1929-2002), destacando a temática da linguagem, sobretudo das linguagens artísticas. A partir dessas expressões, as colocações críticas de Bornheim a respeito da estética e filosofia da arte apresentam um panorama dos questionamentos. Nesse sentido, são notáveis em seus trabalhos as reflexões sobre o teatro e a música. A linguagem teatral permite o acesso às outras atividades artísticas (poesia, música, artes plásticas, cinema) de forma livre e aberta. A linguagem musical, em consonância com a teatral, corrobora a pesquisa de Bornheim, que observou o processo criativo, a comunicação, o papel da interpretação (advento da crítica) e as rupturas nas poéticas contemporâneas. Tal itinerário sublinha a pesquisa que Bornheim realizou na França nas décadas de 1950 e 1960-70. O estímulo dessa atmosfera, marcada pelo diálogo entre filosofia, ciências sociais, psicologia, psicanálise, história, antropologia, linguística, comunicação, teatro e música foi decisivo para ele. Esses pontos são importantes para a apreensão do tema da linguagem e sua ambiência histórica, na qual Bornheim revela outras perspectivas de pesquisa. O pano de fundo é a crise da metafísica e os novos parâmetros para se pensar a dialética, a teoria e a prática. O diagnóstico de tal crise estende-se também à estética. Por conseguinte, o entendimento das ideias de Bornheim conduz aos temas da diferença e alteridade na contemporaneidade. Com isso, persegue-se um percurso temático que aborda: a linguagem e o problema da comunicação a partir da ligação das interpretações de Bornheim com as de Sartre e Merleau-Ponty. Além disso, o surgimento da crítica e os questionamentos da normatividade ética e estética levam à discussão das motivações coincidentes entre artes e ciências. Por fim, a linguagem musical enfatiza ainda o processo de transformação da subjetividade, que propicia uma percepção mais ampla das expressões artísticas e culturais.

Kires: a data-centric telerehabilitation system based on kinect

185 p.

A ecologia em miniatura: o menor tetrápodo do Brasil, Brachycephalus didactylus (Izecksohn, 1971) (Anura: Brachycephalidae) em uma área de Mata Atlântica no Espírito Santo, Sudeste do Brasil

O processo de miniaturização em anuros pode ter implicações ecológicas como, por exemplo, a redução no tamanho da ninhada produzida pelas fêmeas, o aumento do tamanho relativo dos ovos e o desenvolvimento direto. O consequente reduzido tamanho da boca tende também a constituir um fator limitante no consumo de presas por estes pequenos anuros, tornando acessível ao consumo apenas uma gama restrita de presas diminutas. Assim, estudamos parâmetros da ecologia de uma população do anuro Brachycephalus didactylus, em uma área de Mata Atlântica no sul do Espírito Santo, objetivando ampliar o conhecimento sobre tais parâmetros que compreenderam o dimorfismo sexual, algumas características da reprodução, a dieta e a relação com a disponibilidade de atrópodos no folhiço e uso do microhabitat. A amostragem dos dados ocorreu durante um ano, nas estações seca e chuvosa. Brachycephalus didactylus possuiu dimorfismo sexual, com fêmeas sendo maiores do que os machos. O maior tamanho atingido pelas fêmeas provavelmente resulta da seleção intrassexual atuando nas fêmeas para favorecer um maior investimento na massa dos ovos, já que a espécie possui um tamanho de ninhada consideravelmente reduzido (um a dois ovos). A dieta de B. didactylus foi composta por um espectro relativamente amplo de presas (17 itens), tendo elevadas proporções de pequenas presas como Acari e Collembola. O consumo de Formicidae (formigas) foi evitado por B. didactylus, apesar de essa presa ser a mais abundante no folhiço, o que categorizou o anuro como um "especialista em não comer formigas" ("non-ant specialist", sensu Toft 1980a). O reduzido tamanho da boca nas fêmeas de B. didactylus limitou o tamanho máximo de presas passíveis de serem ingeridas pelo anuro e restringindo-o ao consumo de presas pequenas, com maiores indivíduos consumindo um número maior de presas. Por fim, B. didactylus apresentou uma utilização essencialmente horizontal do hábitat, sendo mais frequentemente encontrado sobre o folhiço no chão da mata.

Papel do bloqueio dos receptores NMDA e da ativação dos receptores GABAA na manifestação dos déficits neurocomportamentais decorrentes da exposição ao etanol durante o período equivalente ao terceiro trimestre de gestação

A exposição gestacional ao etanol produz um amplo espectro de defeitos neurocomportamentais que podem persistir ao longo da vida. Dentre os distúrbios mais comumente observados estão o transtorno do déficit de atenção e hiperatividade (TDAH) e os déficits de aprendizado e memória. Apesar da grande quantidade de estudos, os mecanismos envolvidos com a manifestação destes transtornos permanecem pouco conhecidos. Estudos em roedores vêm demonstrando que o período equivalente ao terceiro trimestre de gestação é critico para o aparecimento destas alterações comportamentais. Durante este período, que é caracterizado por intensa sinaptogênese, a neurotoxicidade do etanol vem sendo atribuída ao bloqueio dos receptores glutamatérgicos do tipo N-metil-D-aspartato (NMDA) e hiperativação dos receptores do ácido gama-aminobutírico do subtipo A (GABAA). Tendo em vista que ao longo do desenvolvimento estes receptores diferem em relação a função e distribuição espaço-temporal, neste estudo avaliamos a contribuição relativa do bloqueio dos receptores NMDA e hiperativação dos receptores GABAA durante o período equivalente ao terceiro trimestre de gestação para a manifestação da hiperatividade locomotora e para os distúrbios de aprendizado e memória de camundongos pré-púberes. Para tanto, este estudo foi realizado em duas etapas. Na primeira, investigamos os efeitos da exposição isolada ao bloqueador NMDA MK801 (MK) e ao agonista GABAA muscimol (MU). Para tanto, em dias alternados do segundo dia pós-natal (PN2) a PN8, os animais receberam uma injeção intraperitoneal de Salina (SAL), MK nas doses de 0,1, 0,3 ou 0,5 mg/kg ou de MU nas doses de 0,1 0,3 ou 0,5 mg/kg. Na segunda etapa investigamos os efeitos da administração simultânea de MK (0,1mg/kg) e MU (doses 0,02, 0,1 ou 0,5 mg/kg). Em PN25, a atividade locomotora foi automaticamente avaliada por 15 min no teste de campo aberto. Em PN31 e PN32, o aprendizado e memória foi avaliado no teste da esquiva passiva inibitória. Em relação aos resultados da exposição isolada a cada uma das drogas, apenas o tratamento com MK promoveu um aumento dose dependente na atividade locomotora. No teste da esquiva passiva inibitória, os animais expostos as maiores doses de MK e MU apresentaram déficits de aprendizado e memória. Em relação aos resultados da exposição combinada de MK e MU, não foram observadas diferenças significativas entre os grupos na atividade locomotora. Na esquiva passiva inibitória, a administração simultânea de MK e MU, em doses que administradas isoladamente não tiveram efeito, promoveu prejuízos de aprendizado e memória. Nossos resultados sugerem que, enquanto a hiperatividade locomotora está associada apenas com o bloqueio dos receptores NMDA, os déficits de aprendizado e memória podem ser produto de uma ação sinergista do etanol nos dois receptores.

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。