青蒿开花与青蒿素生物合成相关性的研究-金丝桃和百金花二苯甲酮合酶基因的克隆，异源表达及功能分析

第一部分：青蒿开花与青蒿素生物合成相关性的研究 青蒿素是从中药青蒿中分离出的倍半萜内酯化合物，目前是世界上唯一有效的治疗脑型疟疾和抗氯喹恶性疟疾的药物。青蒿植株中青蒿素含量在开花期最高，但是目前尚不清楚开花与青蒿素生物合成的关系。为此，我们用光周期（短日照）诱导青蒿提前开花，不仅同时获得了开花与不开花的青蒿植株，而且还成功地在同一植株上诱导部分分枝开花，另一部分分枝保持营养生长状态。这一实验体系为研究青蒿开花与青蒿素生物合成的相关性奠定了基础。实验结果表明，开花与不开花青蒿植株青蒿素含量有明显差异。开花植株的青蒿素含量在前2周内逐渐提高，第三周（开花期）达到最高，并保持一周左右，在随后的2周内下降。青蒿植株开花后，叶片便开始老化变黄，逐渐死亡。未开花青蒿植株的青蒿素含量动态在前三周内与开花植株类似，但是这种高青蒿素含量状态能保持较长时间，至少在随后的2周内没有下降。未开花植株的叶片依然保持绿色。这一结果表明，开花不是导致青蒿素含量提高的直接原因。 扫描电镜观察结果表明，幼嫩叶片上的毛状腺体( trichrome)结构是完整的，而在老化的叶片上，则观察到了相当比例(40-50%)破损的腺体。这可能是导致青蒿素含量下降的直接原因。 不同生态型青蒿对光周期的反应是不同的。在北京地区，本地青蒿在8月初便开始开花，而来自四川武陵的青蒿则要到9月份才能开花。根据这一特性，采用“南蒿北栽”的方法，能够使青蒿保持较长时间的营养生长状态，延长适于采收的时间。 第二部分：金丝桃和百金花二苯甲酮合酶基因的克隆，异源表达及功能分析 植物次生代谢物山屯酮( Xanthones)仅存在于龙胆科和藤黄科植物中。它们具有抑制单胺氧化酶，细胞毒素及抗肿瘤活性。 含有1 3个碳原子的二苯甲酮是山屯酮生物合成的中间产物，是由二苯甲酮合酶催化合成的，这一反应是山屯酮生物合成的关键步骤。二苯甲酮合酶已经在金丝桃和百金花细胞悬浮培养系统中检测到，并进行了细致的生化水平上的研究。本研究是在上述研究的基础上，进一步克隆该酶的基因，并进行异源表达及功能分析工作，以便更好地了解和调控山屯酮的生物合成。 用PCR和RT-PCR技术，从金丝桃cDNA文库和逆转录产物中分别克隆到一个基因HBPS1和HBPS2，从百金花cDNA文库中克隆到一个基因CBPS1。HBPS1含有1402个碱基，其开放阅读框架编码390个氨基酸，分子量为42.7 kDa，等电点为6.55。HBPS2含有1398个碱基，其开放阅读框架编码395个氨基酸，分子量为42.8 kDa，等电点为5.78。CBPS1含有1383个碱基，其开放阅读框架编码389个氨基酸，分子量为42.7 kDa，等电点为7.88。与GenBank中序列同源性比较结果表明：在氨基酸水平上，HBPS1与茶（Camellia sinensis）查尔酮合酶的同源性高达92%，HBPS2与萝卜（Raphanus sativus）查尔酮合酶的同源性为64%，CBPS1与茶（Camellia sinensis）查尔酮合酶的同源性为71%。HBPS1与HBPS2的同源性仅为62%。 将三个新克隆的基因的ORF整合到载体pGEX-G上的谷胱甘肽还原酶S基因下游，构建成转化质粒，并在大肠杆菌中诱导表达。结果表明，这三个基因的ORF片段均能被表达成约68 kDa的产物，这与期望的结果一致。 活性检测结果表明，HBPS1是查尔酮合成酶，其底物为香豆酰辅酶A和丙二酸单酰辅酶A，对这两种底物的亲和性KM分别为：香豆酰辅酶A 2.8μM，丙二酸单酰辅酶A，11.2μM。最适反应条件是350C，pH7.0，DTT浓度10 μM。 HBPS2是二苯甲酮合酶，其底物是苯甲丙氨酰辅酶A，和丙二酸单酰辅酶A，对这两种底物的亲和性KM分别为：苯甲丙氨酰辅酶A 2.4 μM，丙二酸单酰辅酶A 9.6μM。最适反应条件是350C，pH 6.5，DTT浓度50 μM。而CBPS1则没有检测到任何活性。从同一种植物中同时获得了查尔酮合酶和二苯甲酮合酶，对研究这两种十分相近的酶的差异表达，酶促反应机制等问题将非常有利。

Urban and peri-urban agricultural production along railway tracks: a case study from the Mumbai Metropolitan Region

Urban and peri-urban agriculture (UPA) contributes to food security, serves as an opportunity for income generation, and provides recreational services to urban citizens. With a population of 21 Million people, of which 60 % live in slums, UPA activities can play a crucial role in supporting people’s livelihoods in Mumbai Metropolitan Region (MMR). This study was conducted to characterize the railway gardens, determine their role in UPA production, and assess potential risks. It comprises a baseline survey among 38 railway gardeners across MMR characterized by different demographic, socio-economic, migratory, and labour characteristics. Soil, irrigation water, and plant samples were analyzed for nutrients, heavy metals, and microbial load. All the railway gardeners practiced agriculture as a primary source of income and cultivated seasonal vegetables such as lady’s finger (Abelmoschus esculentus L. Moench), spinach (Spinacia oleracea L.), red amaranth (Amaranthus cruentus L.), and white radish (Raphanus sativus var. longipinnatus) which were irrigated with waste water. This irrigation water was loaded with 7–28 mg N l^(−1), 0.3–7 mg P l^(−1), and 8–32 mg K l^(−1), but also contained heavy metals such as lead (0.02–0.06 mg Pb l^(−1)), cadmium (0.03–0.17 mg Cd l^(−1)), mercury (0.001–0.005 mg Hg l^(−1)), and pathogens such as Escherichia coli (1,100 most probable number per 100 ml). Levels of heavy metals exceeded the critical thresholds in surface soils (Cr, Ni, and Sr) and produce (Pb, Cd, and Sr). The railway garden production systems can substantially foster employment and reduce economic deprivation of urban poor particularly slum dwellers and migrant people. However this production system may also cause possible health risks to producers and consumers.

In Vitro Activity of the Antifungal Plant Defensin RsAFP2 against Candida Isolates and Its In Vivo Efficacy in Prophylactic Murine Models of Candidiasis

We show that RsAFP2, a plant defensin that interacts with fungal glucosylceramides, is active against Candida albicans, inhibits to a lesser extent other Candida species, and is nontoxic to mammalian cells. Moreover, glucosylceramide levels in Candida species correlate with RsAFP2 sensitivity. We found RsAFP2 prophylactically effective against murine candidiasis.

Reprodução de Meloidogyne javanica em olerícolas e em plantas utilizadas na adubação verde

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Desempenho produtivo da tilápia-do-nilo arraçoada com dieta contendo farelo de nabo forrageiro

Alternative protein sources have been studied in order to reduce feed costs. In a completely random experimental design (five treatments and six replicates) 180 Nile tilapia fingerlings were distributed in 30 aquaria (50 L). The fish were fed isoprotein (28% digestible protein) and isoenergetic (3000 kcal digestible energy kg(-1)) diets where soybean meal was substituted by fodder radish meal at the following levels: 0, 12.5, 25, 50 and 75% Performance and chemical composition of the fillet were determined after 90 days. It was conclude that fodder radish meal can substitute soybean meal up to 25% without any interference on performance and fillet composition of Nile tilapia fingerlings.

Productivity of lettuce and radish cultivations as a function of spacing and of time of establishment of intercropping

The experiment was conducted at UNESP, Jaboticabal-SP, during the period of September to November of 2000, with the objective of evaluating the productivity of the cultivation of lettuce and radishes as a function of spacing between plants and of the time of establishment of intercropping. The experimental design was a completely randomized blocks and four replications. The 14 treatments consisted of combinations of spacing between lines (0.30 and 0.40 m), cultivation systems (intercropping and monoculture), and time of sowing of radish seeds to establish intercropping (0, 7 and 14 days after transplant of lettuce). The cultivars of lettuce and radish were, 'Tainá' and 'Crimson Gigante', respectively. A greater yield of commercial radish roots was obtained with intercropping cultivation. The fresh mass of lettuce in monoculture did not differ from that produced with intercropping. These results suggest that intercropping cultivation between these species is advantageous.

Phytotoxicity of organic extracts of Turnera ulmifolia L. and Turnera diffusa Willd. ex Schult. in cucumber seeds

We evaluated the phytotoxic effects that the hexane, ethyl acetate and methanol extracts from leaves and branches of the species Turnera ulmifolia L. and Turnera diffusa Willd. ex Schult. (Turneraceae), at concentrations of 1.25, 2.50, 3.75 and 5.00 mg ml-1, have on seed germination and seedling development in cucumber (Cucumis sativus L.). None of the extracts tested prevented germination, although the ethyl acetate extracts of T. diffusa, at 3.75 and 5.00 mg ml-1, reduced the mean germination speed and time to germination. Hexane extracts of both species reduced the main root length, number of secondary roots and hypocotyl length. In the ethyl acetate and methanol extracts, the number of secondary roots and hypocotyl length varied by species and concentration. Ethyl acetate extracts of T. ulmifolia at ≤ 2.50 mg ml-1stimulated growth of the main root and hypocotyl, as did T. ulmifolia methanol extracts at 1.25 and 3.75 mg ml-1; all other extract/concentration combinations had an inhibitory effect on those parameters. The hexane and ethyl acetate extracts of T. diffusa inhibited the formation of secondary roots and of the main root, although significant inhibition of hypocotyl growth was observed only at ≥ 2.50 mg ml-1. Comparatively, T. diffusa extracts inhibited development to a greater degree, thus presenting greater allelopathic potential, than did T. ulmifolia extracts.

Influência da compactação do solo em subsuperfície sobre o crescimento aéreo e radicular de plantas de adubação verde de inverno

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Avaliação da sensibilidade de organismos aquáticos e terrestres ao triclopyr

Pós-graduação em Agronomia (Produção Vegetal) - FCAV

Resistence of radish biotypes to iodosulfuron and alternative control.

2016

Resistance of Brassicaceae plants to root-knot nematode (Meloidogyne spp.) in northern Australia

Brassicaceae plants have the potential as part of an integrated approach to replace fumigant nematicides, providing the biofumigation response following their incorporation is not offset by reproduction of plant-parasitic nematodes on their roots. Forty-three Brassicaceae cultivars were screened in a pot trial for their ability to reduce reproduction of three root-knot nematode isolates from north Queensland, Australia: M. arenaria (NQ1), M. javanica (NQ2) and M. arenaria race 2 (NQ5/7). No cultivar was found to consistently reduce nematode reproduction relative to forage sorghum, the current industry standard, although a commercial fodder radish (Raphanus sativus) and a white mustard (Sinapis alba) line were consistently as resistant to the formation of galls as forage sorghum. A second pot trial screened five commercially available Brassicaceae cultivars, selected for their biofumigation potential, for resistance to two nematode species, M. javanica (NQ2) and M. arenaria (NQ5/7). The fodder radish cv. Weedcheck, was found to be as resistant as forage sorghum to nematode reproduction. A multivariate cluster analysis using the resistance measurements, gall index, nematode number per g of root and multiplication for two nematode species (NQ2 and NQ5/7) confirmed the similarity in resistance between the radish cultivar and forage sorghum. A field trial confirmed the resistance of the fodder radish cv. Weedcheck, with a similar reduction in the number of Meloidogyne spp. juveniles recovered from the roots 8 weeks after planting. The use of fodder radish cultivars as biofumigation crops to manage root-knot nematodes in tropical vegetable production systems deserves further investigation.

Isolation and Estimation of Cytokinins and Cytokinin-Containing Transfer RNAs from Cucumis

N6-({Delta}2-Isopentenyl) adenosine antibodies were used for the isolation of free cytokinins and cytokinin-containing tRNAs from parts of Cucumis sativus L. var. Guntur seedlings and for the estimation of cytokinins in them. Immobilized N6-({Delta}2-isopentenyl) adenosine antibodies retained tRNAs containing N6-({Delta}2-isopentenyl) adenosine and N6-(4-hydroxy-3-methylbut-2-enyl) adenosine with equal efficiencies. There were at least five cytokinins in the free form in cucumber seedlings. N6-(4-Hydroxy-3-methylbut-2-enyl) adenosine, N6-({Delta}2-isopentenyl) adenosine, and N6-({Delta}2-isopentenyl) adenine were present at least to the extent of 80, 23, and 9 nanograms, respectively, in the cotyledons and 40, 6, and 3 nanograms, respectively, in the decotyledonated seedlings per gram of tissue. Only two cytokinins were found in the tRNAs of cucumber cotyledons, namely N6-({Delta}2-isopentenyl) adenosine and N6-(4-hydroxy-3-methylbut-2-enyl) adenosine in amounts of 12 and 318 nanograms, respectively, per gram of tissue. Immunoaffinity chromatographic analysis of radiolabeled aminoacyl tRNAs from cucumber cotyledons showed that tRNAPhe and tRNATyr contained cytokinins whereas tRNAAla did not.

First report of Pythiogeton ramosum (Pythiales) in Australia

In 2012, a project was initiated to assess if the soft rot disease of ginger in Australian fields was associated with pathogens other than Pythium myriotylum. Together with nine Pythium spp., ten isolates of a Pythium-like organism were also recovered from ginger with soft rot symptoms. These Pythium-like isolates were identified as Pythiogeton (Py.) ramosum based on its morphology and ITS sequences. In-vitro pathogenicity tests allowed confirmation of pathogenicity of Py. ramosum on excised carrot (Daucus carota), sweet potato (Ipomoea batatas) and potato (Solanum tubersum) tubers, although it was not pathogenic on excised ginger (Zingiber officinale) and radish (Raphanus sativus) rhizome/roots. In addition it was found to be pathogenic on bean (Phaseolus vulgaris), capsicum (Capsicum annuum) and cauliflower (Brassica oleracea var. botrytis) seedlings. This is the first record of Py. ramosum and its pathogenicity in Australia.

Absence of parallelism between polyamine and nucleic acid contents during induced growth of cucumber cotyledons

The cytokinins (benzyladenine or benzyladenosine) decreased spermidine and spermine contents despite increasing putrescine content, when administered to isolated cotyledons of Cucumis sativus L. var. Guntur in organ culture. KCl decreased putrescine contents, although marginally increasing polyamine contents. The cytokinins and/or KCl augmented nucleic acid biosynthesis and accumulation, resulting in enhanced growth and differentiation of the isolated cotyledons. These observations show that polyamine accumulation and growth are not always coupled.

Avaliação de condicionadores em solo para uso em telhados verdes com vistas à retenção hídrica.

As inundações são fenômenos naturais que ocorrem devido às chuvas de grande magnitude, agravadas nas áreas urbanas pela impermeabilização do solo e ineficiência dos sistemas de drenagem. Os telhados verdes surgem como uma medida compensatória estrutural que pode reter parte da água precipitada, adiando o pico de escoamento. O objetivo deste trabalho foi de desenvolver uma combinação de solo e condicionadores para telhado verde, promovam um aumento relevante na capacidade de retenção hídrica e um maior adiamento do pico de escoamento das águas pluviais. Este estudo foi dividido em duas etapas. Na Etapa 1, foram analisadas, em colunas de percolação, três condicionadores nas seguintes concentrações: Gel retentor Stockosorb (2; 4; 6 g/dm3), Fertilizante de liberação lenta Osmocote (4,7; 7,1; 9,0 g/dm3) e Zeólita (30; 50; 70 g/dm3), em três eventos (regas) consecutivos de chuva simulada na intensidade de 57 mm/h. A avaliação das concentrações mais adequadas dentre as testadas para cada condicionante foi baseada nas análises referentes à retenção hídrica (altura do meio após a rega; tempo de adiamento do escoamento; mm retidos; intensidade da água percolada em mm/min) e à qualidade da água percolada (pH, oxigênio dissolvido, turbidez e sólidos). Na Etapa 2, foi avaliada, em vasos, a influência da presença de três espécies de plantas (Arachis pintoi; Raphanus sativus; Lavandula angustifolia) em dois tipos de meio: solo sem condicionadores; solo com condicionadores nas melhores concentrações indicadas na Etapa 1, sendo simulada apenas um evento de chuva de 57 mm/h. Foram analisados parâmetros biológicos (germinação; plantas sobreviventes; comprimento do caule e da raiz; pesos da biomassa do caule e da raiz); retenção hídrica (altura do meio após a rega; tempo de adiamento do escoamento; mm retidos; intensidade da água percolada em mm/min); qualidade da água percolada (pH; oxigênio dissolvido; turbidez; sólidos; nitrato; amônia; fósforo total). Os resultados da Etapa 1 indicaram que o gel promoveu de forma significativa um aumento na retenção hídrica, e adiou o início da percolação de água, além de promover ligeira elevação do pH na água percolada. A adição de zeólita resultou em um aumento significativo da retenção hídrica, porém tal aumento não é vantajoso visto que este representa um custo adicional que poderia ser reduzido com o aumento da proporção do gel na coluna. A adição de fertilizantes não promoveu mudanças na qualidade da água percolada. Na Etapa 2, somente o efeito da presença de A. pintoi (maior produção de biomassa de raiz e caule) e R. sativus foram avaliadas. A presença dos condicionantes no solo proporcionou um desempenho significativamente superior em relação a retenção hídrica (altura do substrato e adiamento do pico de chuva) e qualidade da água percolada (pH e turbidez) quando comparados aos testes realizados na presença somente de solo. O fertilizante influenciou nas altas concentrações dos nutrientes (nitrogênio e fósforo) na água percolado nos resultados. A presença do gel no substrato, resultou numa capacidade superior de retenção de hídrica, e consequentemente no adiamento do pico de intensidade de chuva. Sendo assim, recomenda-se a aplicação do gel em telhados verdes para futuros estudos em ambientes externos.