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。

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.

变水处理与短期遮荫对作物水分利用效率的影响

变水处理与短期遮荫对棉花水分利用效率的影响研究表明 ,在充分供水 -水分胁迫 -复水的变水处理过程中 ,短期遮荫使净光合速率、蒸腾速率、水分利用效率降低 ;光照 -遮荫 -光照期间 ,总的水分利用效率以水分胁迫的最高 ,充分供水的次之 ,复水处理最低 ,其中遮荫 40 %的水分利用效率高于遮荫 75%的处理 .回归分析表明 ,水分利用效率与光合速率表现出极显著正相关 ,两者回归直线的斜率随水分胁迫而增大 ,而水分利用效率与蒸腾速率的关系较弱 ,多数处理两者相关达不到显著水平

土壤水分与短期遮光对棉花光合及其气孔响应的影响

以陆地棉 (Gossypium hirsutum L.Zhongmain No.2 3)为供试材料 ,探讨了在充分供水-水分胁迫 -复水的处理过程中 ,短期不同遮光水平对棉花光合特性及其气孔响应的影响。结果表明 ,在水分处理过程中 ,所有不同遮光水平的棉花叶片对短期遮光具有相似的基本响应规律 :短期遮光使净光合速率迅速降低 ,气孔导度减少 ,但减少速率缓慢 ;遮阳网去掉后 ,叶片气孔重新开放速率和光合恢复被延迟。水分胁迫期间 ,所有遮光处理的水分利用效率均最高 ,但净光合速率均较低 ,光合及其气孔响应受到严重限制 ,遮光 75%受到的影响大于遮光 40 %的处理。复水后水分胁迫带来的残留影响仍然存在 ,结果使水分利用效率在整个水分处理过程中最低 ,遮光 75%的处理尤为明显。上述表明 ,土壤水分与短期遮光对棉花光合及其气孔响应的影响十分显著

Ocorrência de Fusarium oxysporum f. sp. lycopersici raça 3 em tomate no Brasil e seleção de novas fontes de resistência ao patógeno.

A murcha-de-fusário, causada por Fusarium oxysporum f. sp. lycopersici (FOL), é uma importante doença do tomateiro (Lycopersicon esculentum Mill.) no mundo. Existem três raças identificadas do patógeno, sendo que a raça 3 ainda não havia sido registrada no Brasil. Este trabalho teve dois objetivos: comunicar a presença da raça 3 de FOL no Brasil e selecionar fontes de resistência às três raças do patógeno. Nove isolados de FOL foram obtidos de dois híbridos de tomate (Carmen e Alambra) com sintomas de mrucha, provenientes de três lavouras localizadas nos municípios de Venda Nova do Imigrante - Espírito Santo e Domingos Martins 9ES). Estes dois híbridos comerciais de tomate são considerados resistentes ás raças 1 e 2 de FOL. O teste de virulência foi feito com as cultivares: Ponderosa (suscetível a todasa as raças), IPA-5 (resistente à raça 1), Floradade (resistente às raças 1 e 2) e BHRS-2,3 (resistente às raças 1, 2 e 3). Todos os isolados foram virulentos às cultivares Ponderosa, IPA-R e Floradade e ainda infectaram algumas plantas de BHRS-2,3. O teste de virulência foi repetido com as mesmas cultivares mas também incluindo o acesso 'LA 716' da espécie selvagem L. pennellii. Foram obtidos resultados semelhantes para as cultivares, enquanto L. pennellii apresentou uma reação de imunidade ao patógeno. Estes resultadaos comprovam que os novos isolados de ES pertencem à raça 3 de FOL. Uma coleção de germoplasma de acessos de Lycopersicon spp. da Embrapa Hortaliças foi inicialmente avaliada quanto à reação de um dos isolados da raça 3 e uma parte deles às raças 1 e 2. Novas fontes de resistência múltipla foram identificadas em acessos de L. chilense, l. hirsutum e L. peruvianum, sendo dez genótipos imunes às raças 2 e 3 e cinco às três raças. A identificação destas fontes de resistência peermite que os programas de melhoramento de tomate antecipem potenciais problemas, inclusive a emergência de novas raças de FOL, além das raças 1, 2 e 3.

Resistência de acessos de Lycopersicon a Stemphylium solani e S. lycopersici.

A mancha-de-estenfílio, causada por Stemphylium solani e/ou S. lycopersici, é uma conhecida doença do tomateiro que voltou a ser importante, principalmente porque os atuais híbridos plantados no país não apresentam resistência. O controle da mancha-de-estenfílio se baseia no uso de cultivares resistentes ou outras medidas de manejo como o controle químico e a rotação de cultura. Este trabalho teve como objetivo selecionar novas fontes de resistência à doença in Lycopersicon. Entre os acessos de Lycopersicon avaliados para resistência 18 (54,5%) comportaram-se como suscetíveis, três (9,1%) comportaram-se como intermediários e 12 (36,4%) foram resistentes para as duas espécies fúngicas. Todos os acessos resistentes a S. solani também foram resistentes a S. lycopersici. Foram identificados acessos resistentes e intermediários nas espécies L. esculentum (muito provavelmente portando o gene Sm), L. peruvianum e L. hirsutum.

Microscopia eletrônica de varredura da interação Alternaria solani - tomateiro suscetível e resistente.

Objetivando analisar os eventos iniciais das interações A. solani - L. hirsutum var. glabratum e A. solani - L. esculentum ev. Miller, será realizado um trabalho utilizando processamento para microscopia eletrônica de varredura.

Bt-cotton boosts the gross margin of small-scale cotton producers in South Africa

This paper explores some of the issues involved in the Genetic Modification (GM) debate by focusing on one crop that has been modified for pest resistance, cotton (Gossypium hirsutum), and commercially released to small-scale farmers in the Makhathini Flats, KwaZulu Natal, the Republic of South Africa. This was the first commercial release of a GM variety (Bt-cotton) in Sub-Saharan Africa, and thus provides valuable and timely insights into some of the potential advantages and disadvantages of the technology for small-scale farmers in Africa. Even though there are wider concerns regarding the vulnerability of small-scale farmers in the area, the survey results suggest that Bt-cotton generated higher yields and gross margins than non-Bt-cotton. In addition, Bt-cotton significantly reduced the use of pesticide with consequent potential benefits to human health and the environment.

Comparative effect of root-knot nematode on severity of Verticillium and Fusarium wilt in cotton

The effect of root-knot nematode (RKN) (Meloidogyne incognita) on Verticillium dahliae and Fusarium oxysporum f.sp. vasinfectum in cotton (Gossypium hirsutum) was investigated. Two different inoculation methods were used, one in which inoculum was added to the soil, so that nematode and fungal inoculum were in close proximity; the other, inoculation into the stem, whereby the two inocula were spatially separated. Invasion of the roots by RKN enhanced disease severity, as measured by the height of vascular browning in the stem, following inoculation with either wilt pathogen. The effect of RKN on Fusarium wilt was more pronounced than that on Verticillium wilt. Nematode-enhanced infection by F. oxysporum is a well known effect but there are few reports of enhanced infection by Verticillium due to RKN. Relative resistance of a number of cotton cultivars to both wilt diseases, as measured by height of vascular browning, was similar to the known field performance of the cultivars. The use of vascular browning as an estimate of disease severity was therefore validated.

Enhancement of germination of spores of Verticillium dahliae and Fusarium oxysporum esp vasinfectum in vascular fluid from cotton plants infected with the root-knot nematode

Root-knot nematode [RKN] (Meloidogyne incognita) can increase the severity of Verticillium (V dahliae) and Fusarium (F oxysporum f.sp. vasinfectum) wilt diseases in cotton (Gossypium hirsutum). This study was conducted to determine some of the physiological responses caused by nematode invasion that might decrease resistance to vascular wilt diseases. The effect of RKN was investigated on spore germination and protein, carbohydrate and peroxidase content in the xylem fluids extracted from nematode-infected plants. Two cotton cultivars were used with different levels of resistance to both of the wilt pathogens. Spore germination was greater in the xylem fluids from nematode-infected plants than from nematode-free plants. The effect on spore germination was greater in the Fusarium-resistant cultivar (51%). Analysis of these fluids showed a decrease in total protein and carbohydrate levels for both wilt-resistant cultivars, and an increase in peroxidase concentration. Fluids from nematode-free plants of the Verticillium-resistant cultivar contained 46% more peroxidase than the Fusarium-resistant cultivar. The results provide further evidence that the effect of RKN on vascular wilt resistance is systemic and not only local. Changes in metabolites in the xylem pass from the root to the stem, accelerating disease development.

Stability of plasmid profile of highly virulent races of Xanthomonas campestris pv. malvacearum during storage and subculturing

Four races of Xanthomonas campestris pv. mal-vacearum (Xcm) viz. races 23, 27 and 32 (isolated from Gossypium hirsutum) and race 23b (from Gossypium barbadense) were studied. The plasmid profile of the natural isolates showed four plasmids in races 23 and 23b (ca. 60, 40, 23, 8.2 kb), five in race 27 (ca. 60, 40, 23, 8.2 and 3.7 kb) and six in race 32 (ca. 60, 40, 23, 8.2, 3.7 and 1.6 kb). Continuously sub-cultured laboratory isolates of the Xcm races resulted in the loss of all but two plasmids, ca. 60 and 40 kb in size. When the laboratory isolates were passed through cotton (Gossypium hirsutum), they regained certain plasmids so that four plasmids were found in race 23 and 23b (ca. 60, 40, 23 and 8.2 kb), five in race 27 (ca. 60, 40, 23, 8.2 and 3.7 kb) and six in race 32 (ca. 60, 40, 23, 8.2, 3.7 and 1.6 kb), which was more or less similar to the original isolates. The isolates recovered from cotton maintained their plasmid profile (except for minor changes in the miniplasmids) after storage for six months at -70degreesC in 50% glycerol. It is suggested that plasmid profiles among highly virulent races of Xcm are unstable during repeated sub-culturing at room temperature, resulting in rapid loss of some plasmids. However, when the cultures were sub-cultured and stored at -70degreesC the plasmid profile was fairly stable except for the miniplasmids (ca. 3.7 and 1.6 kb).

O gênero geastrum pers. (phallomycetidae, basidiomycota) em algumas áreas de Mata Atlântica e Caatinga no Rio Grande do Norte, Brasil

A taxonomic survey of representatives of the genus Geastrum took place during the rainy season from 2006 to 2009 in four districts in the state of Rio Grande do Norte: Parque Estadual Dunas do Natal (Atlantic Forest), Mata do Jiqui EMPARN (Atlantic Forest) and Estação Ecológica do Seridó (Caatinga). Fourteen species were recorded: G. entomophilum, G. fimbriatum, G. hirsutum, G. javanicum, G. lageniforme, G. lloydianum, G. minimum, G. morganii, G. ovalisporum, G. pectinatum, G. saccatum, G. schweinitzii, G. setiferum and G. triplex. Of these species, eleven occurred in the Atlantic Forest and six in the Caatinga. A new species has been recorded to science, G. entomophilum, other as a first record for Brazil, G. morganii, six new records for the Northeast, and ten new records for Rio Grande do Norte. The material was tumbled in the Herbarium UFRN. Additionally, a survey of the species of the genus deposited in the Herbarium UFRN was accomplished, resulting in 244 herbarium specimens belonging to thirty-three species. Of these ones, twenty-three were collected in Brazil and ten are from Czech Republic, Europe, as donation from the VZ Herbarium

Efeito da densidade de tubérculos de tiririca (Cyperus rotundus) sobre o crescimento inicial de plantas de algodão

O objetivo deste trabalho foi avaliar a interferência de densidades de tubérculos de tiririca no crescimento inicial de plantas de algodão, cultivar Delta Opal. Para isso, utilizaram-se caixas de cimento-amianto (0,60 x 0,60 x 0,25 m) com Latossolo Vermelho Escuro, no centro das quais foram semeadas seis sementes de algodão, em linha, espaçadas de 0,10 m; em seguida, plantaram-se tubérculos de tiririca nas densidades de 0, 5, 10, 15, 25, 50, 75, 100, 125, 150, 175 e 200 tubérculos por caixa, que foram nestas distribuídos aleatoriamente. O delineamento experimental foi o de blocos casualizados, em quatro repetições. Aos 50 dias após a semeadura, foram determinados, nas plantas de algodão, a altura e o teor relativo de clorofila total. Ao término do período experimental (65 dias após a emergência do algodão), foram determinados: altura, teor relativo de clorofila total, área foliar e matéria seca do caule e das folhas. Nas plantas de tiririca foi feita a contagem do número final de plantas (partes aéreas) por caixa. A interferência da tiririca no crescimento inicial do algodoeiro iniciou-se a partir de 5 tubérculos/caixa, reduzindo em até 71% as características analisadas na mais alta densidade (200 tubérculos/caixa). A competição intra-específica na tiririca acentuou-se a partir de 75 tubérculos/caixa, quando obteve mais do que 1,86 brotação por tubérculo. As características avaliadas nas plantas de algodão mais sensíveis ao efeito dos tubérculos de tiririca foram: área foliar, massa seca de folhas, caule e altura das plantas, nesta ordem.

Períodos de interferência das plantas daninhas em algodoeiro de fibra colorida ‘BRS Safira’

This study aimed to determine the late-season presence of weeds in reddish brown cotton (cultivar BRS Safira) and the critical times for removing weeds. The experiment was carried out in the area of Empresa Brasileira de Pesquisa Agropecuária (EMBRAPA) in Missao Velha-CE, Brazil, located at 7 degrees 42'07" S latitude and 39 degrees 24'18" WGr. longitude, during the 2007/2008 season. The treatments consisted of two control groups: weed-free crop during the initial period after crop emergency, and crop kept with weeds during initial period after plant emergency. Both consisted, 0; 20; 40; 60; 80 e 120 days after crop emergency (DAE), the weed community was evaluated by two phytosociological indices (relative dominance and relative importance). Regression analysis was performed as the model of sigmoidal Boltzman, using data from productivity separately within each type of competition, to identify the critical periods of competition, considering 2; 5 and 10% reduction in yield. The community of weeds was composed by 21 species, standing out among them: Richardia grandiflora, Amaranthus deflexus, Eleusine indica, Merremia aegyptia, Eragrotis pilosa, Cenchrus echinatus and Waltheria indica. Considering 2; 5 and 10% reduction in yield fiber as acceptable, the critical period before weed interference were, respectively, 8; 14 and 20 DAE. The critical period of weed interference, for those same levels of losses, were respectively, 100; 82 and 60 days.