小麦Ta-JA1基因功能的初步分析

茉莉酸是植物信号传递以及诱导植物产生防御反应的关键诱导激素之一，广泛存在于高等植物中，并在植物病虫害防御的信号传导通路中起着重要的作用。茉莉酸可以诱导植物抗性基因表达，产生茉莉酸调节蛋白来抵御病虫害。在禾本科的大麦中发现了一个分子量在32kDa茉莉酸调节蛋白家族（JPR-32），但其功能一直没有深入的研究。 木菠萝素是从桂木属木菠萝的种子中分离的一种可以和半乳糖或甘露糖特异结合的凝集素。近来的研究表明，植物的凝集素具有多种功能，主要有：可作为储存蛋白，对储存物质进行包装、运输;作为植物细胞的有丝分裂因子，参与细胞壁的延伸;生长调节及运输碳水化合物;具有酶的功能;参与豆科植物感染结瘤;协同其它防御蛋白参与植物防御反应。植物凝集素的功能复杂各异，对木菠萝素的功能研究更是相对较少。 本文在小麦中克隆出的cDNA（本文命名为Ta-JA1基因），该基因cDNA全长1158bp，编码304个氨基酸，分子量32.7kDa，与JPR-32蛋白质家族的基因序列具有很高的同源性。从蛋白结构分析中显示，Ta-JA1基因有两个典型的功能结构域：N末端的茉莉酸诱导的防御反应结构域和C末端的木菠萝素相关结构域。为我们研究这个新的蛋白家族的功能提供了一个典型的模式蛋白。本文即从Ta-JA1基因出发来研究这一类蛋白的相关功能。在此，我们构建了Ta-JA1基因的pBI121表达载体，并通过农杆菌介导叶圆片法转化烟草，成功获得转基因植株。通过硫酸铵盐析法获得了植物Ta-JA1蛋白粗提品，效率在0.01%左右。使用蛋白粗提物进行凝血效应分析，转基因植株的蛋白粗样品可以凝集新鲜的兔血，说明Ta-JA1蛋白具有植物凝集素的基本性质。选取烟草上典型的三类病原体：烟草花叶病毒，烟草黑胫病菌和烟草野火病菌。分别对转基因烟草进行侵染，并观察统计其抗病性，发现转基因烟草对烟草花叶病毒和烟草黑胫病具有显著的抗性，对烟草野火病也具有一定的抑制作用。通过与野生型烟草在抗盐，抗旱，抗虫和生长发育等方面的统计比较与分析，可以看出，基因烟草在抗逆性上也有了显著的提高，虽然Ta-JA1的过量表达没有影响转基因烟草的整体生长进程，开花期和结实情况与对照烟草相比也无明显变化，但是转基因烟草的种子在萌发时间上有了显著提高，一定数量上还表现出愈合的花冠筒上出现不同程度开裂，花冠筒上有附生舌状花瓣，及带有花瓣状颜色的花萼等异常花表型。

烟草单半乳糖甘油二酯缺失对茉莉酸生物合成的影响

茉莉酸（JA）是由脂肪酸衍生而来的环戊酮化合物，广泛存在于自然界中，在植物逆境胁迫响应和生长发育调节过程中起重要作用。因此，JA被认为是一种新型植物激素。植物JA生物合成的最初底物是三烯脂肪酸（含有三个双键的十八碳和十六碳脂肪酸，18:3和16:3），这些脂肪酸经过脂氧合酶（LOX）、丙二烯氧化物合酶（AOS）和丙二烯氧化物环化酶（AOC）等一系列酶促反应，最终生成JA。JA生物合成所需要的三烯脂肪酸来自叶绿体膜脂。高等植物叶绿体类囊体膜含有四种极性甘油脂，它们是：单半乳糖甘油二酯(MGDG)、双半乳糖甘油二酯(DGDG)、硫代异鼠李糖甘油二酯(SQDG)和磷脂酰甘油(PG)。但是人们尚不清楚JA生物合成所需要的三烯脂肪酸主要来自哪一种膜脂。 最近，我们利用RNA干扰技术获得了烟草MGDG部分缺失的突变体。MGDG是质体中最重要的甘油脂，其含量高达50%，其中含有的三烯脂肪酸约占总脂中三烯脂肪酸含量的65%。本研究的目的是以烟草MGDG缺失的突变体（mgd1）为材料，通过研究MGDG缺失对茉莉酸生物合成的影响，阐明半乳糖脂与JA生物合成的关系。 首先我们对野生型烟草（WT）和mgd1的相关生物学特性进行了研究，包括甘油脂和脂肪酸组成。结果表明，mgd1烟草叶片中MGDG含量降低了57％，同时，其三烯脂肪酸相对含量也大幅度降低。其中十六碳三烯酸（16:3）降低了78%，亚麻酸（18:3）含量减少了28%。因此，由于MGDG缺失，类囊体中的三烯脂肪酸降低了27%。这一结果说明了JA生物合成的底物大幅度减少。 为了说明MGDG缺失导致的三烯脂肪酸含量的减少是否影响到JA的含量，我们利用GC-MS方法比较了WT和mgd1烟草中JA的含量。结果表明，mgd1叶片中的JA含量较WT降低了50%，说明了MGDG的缺失影响了JA的生物合成。 伤害可以诱导JA在短时间内大量合成。我们比较了机械损伤后JA在WT和mgd1叶片中积累的动态过程。伤害同时可以使WT和mgd1叶片中的JA含量增加，并且在1小时达到最大值。但是，JA在两种烟草叶片中增加的幅度不同，WT叶片受伤1小时后JA含量是未受伤时的5倍，而mgd1叶片受伤1小时后，其JA含量只增加了1倍。这些结果说明了MGDG缺失可以严重影响伤害诱导的 JA 的积累，MGDG是JA的生物合成底物的重要来源。 我们进一步研究了MGDG缺失对JA生物合成相关酶基因表达的影响。 LOX1和AOC编码JA生物合成途径中的关键酶LOX和AOC。RT-PCR分析表明mgd1叶片中这两个基因受伤害激活的程度比WT弱。进一步说明突变体中JA合成受到影响。 植物受到伤害时内源JA含量增加，并激活防御基因的表达。我们的结果显示，当植物受伤害后，mgd1叶片中与JA信号转导相关的防御基因HPL，PI-I和PI-II的表达量增加幅度明显低于WT。这说明突变体中JA信号转导途径受到了抑制。 JA在植物对昆虫侵害的防御反应中起重要作用，上述结果表明突变体对伤害响应受到削弱。昆虫饲喂实验显示，棉铃虫更趋向食用mgd1植株叶片，取食mgd1植株的棉铃虫的体重增加较多。这些结果与WT和mgd1在JA含量、防御相关基因表达方面的差异相一致。外源施加茉莉酸甲酯（MeJA）能够恢复mgd1的抗虫性和防御基因的表达，说明JA是恢复mgd1抗虫性所必须的。 上述结果表明MGDG缺失使JA生物合成受到影响，尤其是JA在植物受到伤害后的生物合成。对于这一现象的可能的解释是：MGDG是JA生物合成底物的主要来源，由于mgd1中缺少大量的MGDG，当植物受到伤害时，MGDG不能释放出足够三烯脂肪酸来合成JA，导致其含量降低，破坏了JA信号途径，最终使得植株表现出抗性降低等特性。我们的研究证明了MGDG可以作为JA生物合成的底物来源在JA信号途径中起重要作用。

茉莉酸结构类似物的合成及其对银杏次生代谢的影响

茉莉酸类化合物是一种具有诱导植物次生代谢，抵抗外来侵害，提高植物抗逆防御性等重要作用的植物激素，主要包括茉莉酸（JA）、茉莉酸甲酯（MeJA）及其异亮氨酸衍生物等。茉莉酸的化学合成已报道有十几种之多，但都还无法实现工业化生产，满足农业需求。 本文以α-羧基肉桂酸为起始原料，经过加氢还原、AlCl3-NaCl离子液体中的分子内F-C酰基化反应，HATU催化下与异亮氨酸结合三步反应，合成了一种茉莉酸类似物吲哚异亮氨酸甲酯结合物（Ind-IleMe），总收率约70 %。 用合成的Ind-IleMe、coronalon及MeJA对银杏叶进行诱导，银杏叶经盐酸甲醇溶液加热水解提取黄酮苷元，HPLC检测发现，经诱导后的银杏叶与对照相比银杏黄酮含量均有所增加，最高可诱导银杏叶黄酮含量增长15%—20%，最高诱导浓度coronalon 最低，为1 μmol/L，Ind-IleMe是10 μmol/L，MeJA的诱导浓度最高，大于等于100 μmol/L。 对MeJA诱导后银杏叶的生理生化中几个重要指标进行了测定，发现经诱导后SOD、MDA、PAL、蛋白活性或含量相对升高，叶绿素、可溶性糖含量相对降低，这与MeJA诱导提高银杏叶的逆境防御及次生代谢有关。 在植物的次生代谢中，挥发性气体（VOCs）具有防御草食动物及实现同种及不同种植物间信息交流的重要作用。本文应用自组装的炭阱吸附装置和固相微萃取（SPME）来收集诱导后的银杏叶、利马豆及三种酒中的挥发物，GC-MS检测进行定性定量分析，发现诱导后的银杏叶释放出更多的挥发性有机物，主要是石竹烯等一些参与植物防御机制的倍半萜类，通过对比炭阱吸附和SPME在挥发物的收集上，发现炭阱吸附具有吸附效率更高、样品可短期保存、重复进样分析、可定量等优越性，因此具有很好的发展前景。

两种真核微藻逆境响应关键基因的调控机理研究

非生物逆境胁迫严重影响植物的生长发育和作物的产量。解析逆境下植物体基 因表达及信号传导网络调控机制对于阐明植物的适应性反应及培育耐逆性作物具有 重要理论和现实意义。 雨生红球藻（Haematoccoccus pluvialis）通过累积虾青素来适应胁迫环境，茉莉 酮酸甲酯（methyl jasmonate，MJ）和赤霉素（gibberellins，GA）对植物胁迫适应过 程中相关基因的诱导表达起到重要的调控作用。本文选取 H. pluvialis 作为出发株， 研究了 MJ 和 GA 对虾青素合成途径关键酶 β-胡萝卜素酮化酶基因的调控作用。 在其中一种 H. pluvialis 中克隆获得了三种不同的 β-胡萝卜素酮化酶基因。通过基 因组步移，获得了三种 β-胡萝卜素酮化酶的5’ 侧翼序列（5’-flanking region），其 中存在着多样的顺式作用元件，包括 MJ 和 GA 的顺式作用位点。进一步的实验 表明，MJ 和 GA 的诱导处理，可以促进 H. pluvialis 中虾青素的累积；同时，MJ 和 GA 调控了三种 β-胡萝卜素酮化酶基因的转录水平。 南极小球藻（Chlorella vulgaris NJ-7）可以在较大温度和盐度变化范围内存活， 是研究生物胁迫适应机理的理想模式生物。对数期的 C. vulgaris NJ-7 主要的脂肪酸

Instability of anthocyanin accumulation in Vitis vinifera L. var. Gamay Freaux suspension cultures

The inherent instability of metabolite production in plant cell culture-based bioprocessing is a major problem hindering its commercialization. To understand the extent and causes of this instability, this study was aimed at understanding the variability of anthocyanin accumulation during long-term subcultures, as well as within subculture batches, in Vitis vinifera cell cultures. Therefore, four cell line suspensions of Vitis vinitera L. var. Gamay Freaux, A, B, C and D, originated from the same callus by cell-aggregate cloning, were established with starting anthocyanin contents of 2.73 +/- 0.15, 1.45 +/- 0.04, 0.77 +/- 0.024 and 0.27 +/- 0.04 CV (Color Value)/g-FCW (fresh cell weight), respectively. During weekly subculturing of 33 batches over 8 months, the anthocyanin biosynthetic capacity was gradually lost at various rates, for all four cell lines, regardless of the significant difference in the starting anthocyanin content. Contrary to this general trend, a significant fluctuation in the anthocyanin content was observed, but with an irregular cyclic pattern. The variabilities in the anthocyanin content between the subcultures for the 33 batches, as represented by the variation coefficient (VC), were 58, 57, 54, and 84% for V vinifera cell lines A, B, C and D, respectively. Within one subculture, the VCs from 12 replicate flasks for each of 12 independent subcultures were averaged, and found to be 9.7%, ranging from 4 to 17%. High- and low-producing cell lines, VV05 and VV06, with 1.8-fold differences in their basal anthocyanin contents, exhibited different inducibilities to L-phenylalanine feeding, methyl jasmonate and light irradiation. The low-producing cell line, showed greater potential in enhanced the anthocyanin production.

La régulation génique chez Solanum chacoense : de la pollinisation jusqu’à l’embryogenèse

Chez les végétaux supérieurs, l’embryogenèse est une phase clé du développement au cours de laquelle l’embryon établit les principales structures qui formeront la future plante et synthétise et accumule des réserves définissant le rendement et la qualité nutritionnelle des graines. Ainsi, la compréhension des évènements moléculaires et physiologiques menant à la formation de la graine représente un intérêt agronomique majeur. Toutefois, l'analyse des premiers stades de développement est souvent difficile parce que l'embryon est petit et intégré à l'intérieur du tissu maternel. Solanum chacoense qui présente des fleurs relativement grande facilitant l’isolation des ovules, a été utilisée pour l’étude de la biologie de la reproduction plus précisément la formation des gamètes femelles, la pollinisation, la fécondation et le développement des embryons. Afin d'analyser le programme transcriptionnel induit au cours de la structuration de ces étapes de la reproduction sexuée, nous avons mis à profit un projet de séquençage de 7741 ESTs (6700 unigènes) exprimés dans l’ovule à différents stades du développement embryonnaire. L’ADN de ces ESTs a été utilisé pour la fabrication de biopuces d’ADN. Dans un premier temps, ces biopuces ont été utilisé pour comparer des ADNc issus des ovules de chaque stade de développement embryonnaire (depuis le zygote jusqu’au embryon mature) versus un ovule non fécondé. Trois profils d’expression correspondant au stade précoce, intermédiaire et tardive ont été trouvés. Une analyse plus approfondie entre chaque point étudié (de 0 à 22 jours après pollinisation), a permis d'identifier des gènes spécifiques caractérisant des phases de transition spécifiques. Les annotations Fonctionnelles des gènes differentiellement exprimés nous ont permis d'identifier les principales fonctions cellulaires impliquées à chaque stade de développement, révélant que les embryons sont engagés dans des actifs processus de différenciation. Ces biopuces d’ADN ont été par la suite utilisé pour comparer différent types de pollinisation (compatible, incompatible, semi-compatible et inter-espèce) afin d’identifier les gènes répondants à plusieurs stimuli avant l'arrivé du tube pollinique aux ovules (activation à distance). Nous avons pu démontrer que le signal perçu par l’ovaire était différent et dépend de plusieurs facteurs, incluant le type de pollen et la distance parcourue par le pollen dans le style. Une autre analyse permettant la comparaison des différentes pollinisations et la blessure du style nous a permis d’identifier que les programmes génétiques de la pollinisation chevauchent en partie avec ceux du stress. Cela était confirmé en traitant les fleurs par une hormone de stress, méthyle jasmonate. Dans le dernier chapitre, nous avons utilisé ces biopuces pour étudier le changement transcriptionnel d’un mutant sur exprimant une protéine kinase FRK2 impliqué dans l’identité des ovules. Nous avons pu sélectionner plusieurs gènes candidat touchés par la surexpression de cette kinase pour mieux comprendre la voie se signalisation. Ces biopuces ont ainsi servi à déterminer la variation au niveau transcriptionnelle des gènes impliqués lors de différents stades de la reproduction sexuée chez les plantes et nous a permis de mieux comprendre ces étapes.

Foraging strategies of natural enemies of the peach-potato aphid Myzus persicae

The foraging strategies of two natural enemies of the peach-potato aphid Myzus persicae: the seven-spot ladybird Coccinella septempunctata and the parasitoid wasp Diaeretiella rapae, were investigated. Specifically the roles of plant semiochemicals in the location of plants infested with M. persicae by these natural enemies were examined. I investigated the olfactory responses of female C. septempunctata to volatiles collected from M. persicae and four Brassica cultivars; Brassica rapa, B. juncea, B. napus cultivar ‘Apex’ and B. napus cultivar ‘Courage’ and wild-type Arabidopsis thaliana that were: undamaged, previously infested by M. persicae and infested with M. persicae. C. septempunctata showed no attraction to volatiles from M. persicae alone. C. septempunctata significantly changed its searching behaviour in response to plant volatiles from B. rapa, B. napus cv. ‘Apex’ and Arabidopsis infested with M. persicae. C. septempunctata was also found to display a significant turning bias when foraging on a branching horizontal wire stem. A model was developed to investigate how turning biases affect the foraging efficiency of C. septempunctata in dichotomous branched environments. Simulations using this model indicated that turning biases could potentially increase searching efficiency. D. rapae showed a significant preference for volatiles from M. persicae infested wild-type Arabidopsis but no preference to volatiles from M. persicae alone or M. persicae honeydew. Volatile emissions by Arabidopsis were shown to be localised to the area of aphid-infestation rather than systemic. Using gas chromatography plants infested with M. persicae were shown to emit a quantitatively different volatile blend than undamaged plants. In experiments with jasmonate mutants of Arabidopsis the jasmonate (octadecanoid) wound response pathway was implicated as being important for the production of M. persicae induced volatiles, attractive to D. rapae. Other wound response pathways were also found to be involved in the production of the full blend of M. persicae induced volatiles.

Signal transduction-related responses to phytohormones and environmental challenges in sugarcane

Background: Sugarcane is an increasingly economically and environmentally important C4 grass, used for the production of sugar and bioethanol, a low-carbon emission fuel. Sugarcane originated from crosses of Saccharum species and is noted for its unique capacity to accumulate high amounts of sucrose in its stems. Environmental stresses limit enormously sugarcane productivity worldwide. To investigate transcriptome changes in response to environmental inputs that alter yield we used cDNA microarrays to profile expression of 1,545 genes in plants submitted to drought, phosphate starvation, herbivory and N-2-fixing endophytic bacteria. We also investigated the response to phytohormones (abscisic acid and methyl jasmonate). The arrayed elements correspond mostly to genes involved in signal transduction, hormone biosynthesis, transcription factors, novel genes and genes corresponding to unknown proteins.Results: Adopting an outliers searching method 179 genes with strikingly different expression levels were identified as differentially expressed in at least one of the treatments analysed. Self Organizing Maps were used to cluster the expression profiles of 695 genes that showed a highly correlated expression pattern among replicates. The expression data for 22 genes was evaluated for 36 experimental data points by quantitative RT-PCR indicating a validation rate of 80.5% using three biological experimental replicates. The SUCAST Database was created that provides public access to the data described in this work, linked to tissue expression profiling and the SUCAST gene category and sequence analysis. The SUCAST database also includes a categorization of the sugarcane kinome based on a phylogenetic grouping that included 182 undefined kinases.Conclusion: An extensive study on the sugarcane transcriptome was performed. Sugarcane genes responsive to phytohormones and to challenges sugarcane commonly deals with in the field were identified. Additionally, the protein kinases were annotated based on a phylogenetic approach. The experimental design and statistical analysis applied proved robust to unravel genes associated with a diverse array of conditions attributing novel functions to previously unknown or undefined genes. The data consolidated in the SUCAST database resource can guide further studies and be useful for the development of improved sugarcane varieties.

Análise do perfil de expressão dos genes da cana-de-açúcar envolvidos na interação com Leifsonia xyli subsp: xyli

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

Qualidade de rosas de corte tratadas com produtos naturais

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

Use of Natural Products for Post-Harvest Preservation of 'Vega' Cut Roses

Use of natural products as floral preservatives has helped to reduce the indiscriminate use of chemical products in flower preservation. In this study, we tested the ability of certain natural products to maintain the quality and to increase the commercial durability of 'Vega' cut roses. We employed a randomized factorial design with six post-harvest treatments and four evaluation dates. The following treatments were tested: 1) distilled water; 2) methyl jasmonate (350 mu M) applied in a four-hour pulse; 3) methyl jasmonate (500 mu M) spraying; 4) mint oil (100 ppm); 5) ginger oil (100 ppm); and 6) propolis (0.05%) as a maintenance solution. Flowers were kept at 20+/-2 degrees C and 67+/-3% RH. Physiological and qualitative evaluations were conducted. Natural products had a beneficial effect on the shelf life of the flowers. However, for all evaluated parameters, the methyl jasmonate spray was the most efficient treatment to maintain floral quality, resulting in less fresh-mass loss and a lower flower respiratory rate. Methyl jasmonate spray also improved the maintenance of coloration, relative water content and concentration of reducing sugars, thus extending the shelf life of roses.

Leaf senescence in tomato mutants as affected by irradiance and phytohormones

We explored the interaction between radiation of different wavelength and jasmonic acid (JA) or brassinosteroids (BR) on leaf senescence-induced oxidative stress. Three approaches were used: 1) jasmonic acid insensitive1-1 (jai1-1) and brassinosteroid-deficient [dumpy (dpy)] mutants were treated with red (R) or far-red (FR) radiation; 2) phytochromedeficient aurea (au) and high pigment-1 (hp-1) (radiation exaggerated response) mutants were treated with methyl jasmonate (MeJA) or epibrassinolide (epiBL); and 3) double mutants au jai1-1 and au dpy were produced. Leaf chlorophyll content, lipid peroxidation, and antioxidant enzyme activities were determined. After senescence induction in detached leaves, we verified that the patterns of chlorophyll degradation of hormonal and photomorphogenic mutants were not significantly different in comparison with original cv. Micro-Tom (MT). Moreover, there was no significant change in lipid peroxidation measured as malondialdehyde (MDA) production, as well as catalase (CAT), ascorbate peroxidase (APX), and glutathione reductase (GR) activities in the hormonal mutants. Exogenous BR increased CAT and APX activities in MT, au, and hp-1. As concerns the double mutants, severe reduction in H2O2 production which was not accompanied by changes in MDA content, and CAT and APX activities was observed during senescence in au dpy. The results suggest that JA and BR do not participate in light signaling pathway during leaf senescence-induced oxidative stress. © 2013 Springer Science+Business Media Dordrecht.

Expressão gênica diferencial em palmitos de cana-de-açúcar submetida a diferentes períodos de estresse hídrico

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Avaliação de metil jasmonato na indução de resistência de plantas de Eucalyptus spp. ao psilídeo-de-concha Glycaspis brimblecombei Moore (Hemiptera: Psyllidae

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Aplicação de reguladores vegetais e de fosfito de potássio em mudas de eucalipto submetidas à deficiência hídrica

Pós-graduação em Agronomia (Irrigação e Drenagem) - FCA