花色改变的分子机理研究

自1987年世界首例成功运用转基因技术改造矮牵牛花色以来,花色改造基因工程技术不断展现它在培育新花色品系上的无穷魅力。综述了观赏植物花色素的种类、花色素苷的生物合成途径;关键酶的种类;基因工程改变花色的原理和策略以及花色改良方面的研究进展。     

基因工程技术在花卉中的应用

近年来花卉产业得到了蓬勃发展,不断成熟的基因工程技术为花卉的品质改良提供了新的思路,也为花卉产业化发展带来了新的机遇。本文综述了影响植物花色、花型、花期等相关基因及转基因的研究,展望了基因工程技术在花卉育种和产业化应用的发展前景。     

杨树粒肩天牛的生物学特性

杨树粒肩天牛在福建省2a发生1代;老熟幼虫在第2年4月下旬至5月初开始化蛹,成虫出现期始于5月底6月初,6月中下旬到7月上旬为盛发期,9月上旬只见个别成虫;产卵期始于6月中旬,6月下旬至7月中旬为产卵高峰期;幼虫孵化期为6月下旬到8月中旬,7月上中旬为为盛发期;幼虫不越冬。杨树粒肩天牛成虫为补充营养所取食的树种较集中,主要是构树与桑树。杨树粒肩天牛幼虫对杨树危害严重,但成虫却不喜欢取食杨树,用杨树饲养的粒肩天牛成虫寿命很短,仅3-19d,不产卵;而以桑树为补充营养的雌成虫的平均寿命为55d、雄26.5d;以构树为补充营养的雌成虫的平均寿命为78d、雄45.5d,补充桑树与构树的粒肩天牛成虫均可正常产卵。雌雄成虫一生可多次交尾。雌虫有多次产卵现象,单雌每天产卵数量1-12粒不等。     

Probing the local conformational change of alpha1-antitrypsin

The native form of serpins (serine protease inhibitors) is a metastable conformation, which converts into a more stable form upon complex formation with a target protease. It has been suggested that movement of helix-F (hF) and the following loop connecting to strand 3 of beta-sheet A (thFs3A) is critical for such conformational change. Despite many speculations inferred from analysis of the serpin structure itself, direct experimental evidence for the mobilization of hF/thFs3A during the inhibition process is lacking. To probe the mechanistic role of hF and thFs3A during protease inhibition, a disulfide bond was engineered in alpha(1)-antitrypsin, which would lock the displacement of thFs3A from beta-sheet A. We measured the inhibitory activity of each disulfide-locked mutant and its heat stability against loop-sheet polymerization. Presence of a disulfide between thFs3A and s5A but not between thFs3A and s3A caused loss of the inhibitory activity, suggesting that displacement of hF/thFs3A from strand 5A but not from strand 3A is required during the inhibition process. While showing little influence on the inhibitory activity, the disulfide between thFs3A and s3A retarded loop-sheet polymerization significantly. This successful protein engineering of alpha(1)-antitrypsin is expected to be of value in clinical applications. Based on our current studies, we propose that the reactive-site loop of a serpin glides through between s5A and thFs3A for the full insertion into beta-sheet A while a substantial portion of the interactions between hF and s3A is kept intact.     

Self-assembly of coiled-coil tetramers in the 1.40 A structure of a leucine-zipper mutant

The hydrophobic core of the GCN4 leucine-zipper dimerization domain is formed by a parallel helical association between nonpolar side chains at the a and d positions of the heptad repeat. Here we report a self-assembling coiled-coil array formed by the GCN4-pAe peptide that differs from the wild-type GCN4 leucine zipper by alanine substitutions at three charged e positions. GCN4-pAe is incompletely folded in normal solution conditions yet self-assembles into an antiparallel tetraplex in crystals by formation of unanticipated hydrophobic seams linking the last two heptads of two parallel double-stranded coiled coils. The GCN4-pAe tetramers in the lattice associate laterally through the identical interactions to those in the intramolecular dimer-dimer interface. The van der Waals packing interaction in the solid state controls extended supramolecular assembly of the protein, providing an unusual atomic scale view of a mesostructure.     

A cross-strand Trp Trp pair stabilizes the hPin1 WW domain at the expense of function

Using the human Pin1 WW domain (hPin1 WW), we show that replacement of two nearest neighbor non-hydrogen-bonded residues on adjacent beta-strands with tryptophan (Trp) residues increases beta-sheet thermodynamic stability by 4.8 kJ mol(-1) at physiological temperature. One-dimensional NMR studies confirmed that introduction of the Trp-Trp pair does not globally perturb the structure of the triple-stranded beta-sheet, while circular dichroism studies suggest that the engineered cross-strand Trp-Trp pair adopts a side-chain conformation similar to that first reported for a designed "Trp-zipper" beta-hairpin peptide, wherein the indole side chains stack perpendicular to each other. Even though the mutated side chains in wild-type hPin1 WW are not conserved among WW domains and compose the beta-sheet surface opposite to that responsible for ligand binding, introduction of the cross-strand Trp-Trp pair effectively eliminates hPin1 WW function as assessed by the loss of binding affinity toward a natural peptide ligand. Maximizing both thermodynamic stability and the domain function of hPin1 WW by the above mentioned approach appears to be difficult, analogous to the situation with loop 1 optimization explored previously. That introduction of a non-hydrogen-bonded cross-strand Trp-Trp pair within the hPin1 WW domain eliminates function may provide a rationale for why this energetically favorable pairwise interaction has not yet been identified in WW domains or any other biologically evolved protein with known three-dimensional structure.     

Cell-bionics: tools for real-time sensor processing

The accurate monitoring of the physiological status of cells, tissues and whole organisms demands a new generation of devices capable of providing accurate data in real time with minimal perturbation of the system being measured. To deliver on the promise of cell-bionics advances over the past decade in miniaturization, analogue signal processing, low-power electronics, materials science and protein engineering need to be brought together. In this paper we summarize recent advances in our research that is moving us in this direction. Two areas in particular are highlighted: the exploitation of the physical properties inherent in semiconductor devices to perform very low power on chip signal processing and the use of gene technology to tailor proteins for sensor applications. In the context of engineered tissues, cell-bionics could offer the ability to monitor the precise physiological state of the construct, both during 'manufacture' and post-implantation. Monitoring during manufacture, particularly by embedded devices, would offer quality assurance of the materials components and the fabrication process. Post-implantation monitoring would reveal changes in the underlying physiology as a result of the tissue construct adapting to its new environment.     

Negative plant-soil feedbacks may limit persistence of an invasive tree due to rapid accumulation of soil pathogens

Soil organisms influence plant species coexistence and invasion potential. Plant-soil feedbacks occur when plants change soil community composition such that interactions with that soil community in turn may positively or negatively affect the performance of conspecifics. Theories predict and studies show that invasions may be promoted by stronger negative soil feedbacks for native compared with exotic species. We present a counter-example of a successful invader with strong negative soil feedbacks apparently caused by host-specific, pathogenic soil fungi. Using a feedback experiment in pots, we investigated whether the relative strength of plant-soil feedbacks experienced by a non-native woody invader, Sapium sebiferum, differed from several native tree species by examining their performance in soils collected near conspecifics ('home soils') or heterospecifics ('away soils') in the introduced range. Sapium seedlings, but no native seedlings, had lower survival and biomass in its home soils compared with soils of other species (negative feedback'). To investigate biotic agents potentially responsible for the observed negative feedbacks, we conducted two additional experiments designed to eliminate different soil taxa ('rescue experiments'). We found that soil sterilization (pot experiment ) or soil fungicide applications (pot and field experiments) restored Sapium performance in home soil thereby eliminating the negative feedbacks we observed in the original experiment. Such negative feedbacks apparently mediated by soil fungi could have important effects on persistence of this invader by limiting Sapium seedling success in Sapium dominated forests (home soils) though their weak effects in heterospecific (away) soils suggest a weak role in limiting initial establishment.     

Biological solutions to transport network design

Transport networks are vital components of multicellular organisms, distributing nutrients and removing waste products. Animal and plant transport systems are branching trees whose architecture is linked to universal scaling laws in these organisms. In contrast, many fungi form reticulated mycelia via the branching and fusion of thread-like hyphae that continuously adapt to the environment. Fungal networks have evolved to explore and exploit a patchy environment, rather than ramify through a three-dimensional organism. However, there has been no explicit analysis of the network structures formed, their dynamic behaviour nor how either impact on their ecological function. Using the woodland saprotroph Phanerochaete velutina, we show that fungal networks can display both high transport capacity and robustness to damage. These properties are enhanced as the network grows, while the relative cost of building the network decreases. Thus, mycelia achieve the seemingly competing goals of efficient transport and robustness, with decreasing relative investment, by selective reinforcement and recycling of transport pathways. Fungal networks demonstrate that indeterminate, decentralized systems can yield highly adaptive networks. Understanding how these relatively simple organisms have found effective transport networks through a process of natural selection may inform the design of man-made networks.     

Knockdown of berberine bridge enzyme by RNAi accumulates (<Emphasis Type="Italic">S</Emphasis>)-reticuline and activates a silent pathway in cultured California poppy cells

Reticuline is a key compound in the biosynthetic pathway for isoquinoline alkaloids in plants, which include morphine, codeine and berberine. We established cultured California poppy (Eschscholzia californica) cells, in which berberine bridge enzyme (BBE) was knocked down by RNA interference, to accumulate the important key intermediate reticuline. Both BBE mRNA accumulation and enzyme activity were effectively suppressed in transgenic cells. In these transgenic cells, end-products of isoquinoline alkaloid biosynthesis, such as sanguinarine, were considerably reduced and reticuline was accumulated at a maximum level of 310 μg/g-fresh weight. In addition, 1 g-fresh weight of these cells secreted significant amounts of reticuline into the medium, with a maximum level of 6 mg/20 mL culture medium. These cells also produced a methylated derivative of reticuline, laudanine, which could scarcely be detected in control cells. We discuss the potential application of RNAi technology in metabolic modification and the flexibility of plant secondary metabolism.