植物蛋白质磷酸化的研究技术

本文介绍植物蛋白质磷酸化研究的技术及其应用情况,并对这些技术的应用前景作了展望。     

溴氰菊酯对不同品系家蝇脑突触体膜蛋白磷酸化及ATP酶活性的影响

以敏感品系家蝇和溴氰菊酯抗性品系家蝇(Musca domestica L.)为材料,研究和比较了神经毒剂溴氰菊酯对其脑突触体蛋白磷酸化作用的影响。结果表明,浓度为10^-5 mol/L溴氰菊酯抑制了敏感品系家蝇脑突触体蛋白磷酸化作用,而对抗性品系家蝇脑突触体蛋白磷酸化作用无明显影响。若反应体系中加入2.5×10^-6 mol/L的cAMP显著激活了敏感品系家蝇脑突触体蛋白磷酸化水平,但是当0.6 mmol/Lca²⁺或0.6 mmol/L Ca²⁺加10^-5 mol/L钙调蛋白时明显增强了抗性品系家蝇脑突触体蛋白磷酸化水平,甚至超过了其对敏感品系的作用水平。此外,还发现不同浓度的溴氰菊酯可抑制突触膜上的Na/K-ATP酶和Ca-ATP酶活力,浓度越高抑制作用也越大,并且敏感品系家蝇对溴氰菊酯的敏感度要高于抗性品系。     

用蛋白质组学方法解析磷酸化蛋白质

蛋白质磷酸化和去磷酸化这一可逆过程参与了高等真核生物细胞信号转导、细胞分化和细胞生长等重要过程,并与许多疾病、肿瘤的发生密切相关。蛋白质组学技术的不断发展和完善,可以更好、更多地识别和鉴定磷酸化蛋白质,为解析磷酸化蛋白质提供了可能。章综述了用于分离和鉴定磷酸化蛋白质的蛋白质组学方法。     

吗啡及第二信使对鼠脑细胞膜蛋白质磷酸化的调节

 本文研究了几种蛋白激酶活化剂及吗啡对脑细胞膜蛋白质磷酸化的调节。cAMP刺激了一种68KDa蛋白质和几种60KDa相关的蛋白质的磷酸化作用,Ca~(++)刺激68KDa和50KDa蛋白质的磷酸化。μ吗啡受体的特异性兴奋剂D-脑啡肽(DAGO)增加68KDa蛋白质的磷酸化,而吗啡K受体的特异性兴奋剂,Bremazocyne抑制这一蛋白质的磷酸化。蛋白激酶c的特异性活化剂——磷脂酰丝氨酸(PS)和甘油二油酸酯(DO)不促进这一磷酸化。相反,却抑制cAMP、Ca~(++)、和DAGO所刺激的68KDa蛋白质的磷酸化。结果表明,在鼠脑细胞膜存在一种68KDa专一的蛋白激酶,其活性受吗啡及几种细胞内信使分子,如cAMP、Ca~(++)和DO的调节。     

杨树蛋白质磷酸化位点预测

以小黑杨磷酸化蛋白质组为研究对象,用人工神经网络表达丝氨酸、苏氨酸等残基位点的磷酸化与氨基酸序列的结构特征之间的非线性关系,建立了BP人工神经网络模型,并用磷酸化数据对所建模型进行训练和分析,得适宜的结构为21×16∶8∶4,拟合准确度为90%,Acc、Sn、Sp、MCC分别为78%、89%、67%、0.57,对比分析结果表明,所建模型具有较强的预测能力。     

磷酸化蛋白质组研究中的富集策略

蛋白质的磷酸化与去磷酸化过程,调控着包括信号转换、基因表达、细胞周期等诸多细胞过程。因此,对蛋白质磷酸化修饰的分析是蛋白质组研究中的重要内容。但由于磷酸化蛋白的丰度较低,难以用质谱直接检测。为了解决这个问题,改善质谱对磷酸肽的信号响应,需要对磷酸化蛋白质或磷酸肽进行富集。目前主要的富集方法包括免疫沉淀、固相金属离子亲和色谱、金属氧化物/氢氧化物亲和色谱等。     

小菜蛾溴氰菊酯敏感和抗性品系蛋白质表达谱的比较分析

杀虫剂抗性是害虫防治中的一个主要挑战。为有效治理抗性, 我们必须了解杀虫剂诱导的害虫生理生化变化。目前害虫抗药性的一些机理已经清楚, 但更多的相关机制还有待探究。本研究通过蛋白质组学方法检测了小菜蛾Plutella xylostella溴氰菊酯敏感和抗性品系间蛋白质组的表达差异。结果显示： SDS-PAGE胶上有大约300个蛋白差异点, 其中23个蛋白点具2.5倍以上的表达差异, 通过MALDI-TOF-MS, 我们成功鉴定出8个蛋白, 其中包括化感蛋白CSP2、 铜锌超氧化物歧化酶和peroxiredoxin样蛋白。通过实时定量PCR（real-time quantitative PCR, qPCR）分析了其中5个蛋白的mRNA 表达水平, 结果表明mRNA 表达水平不能真实反映蛋白的表达水平。免疫印迹验证了双向电泳中SOD1的表达差异。本研究有力地证明溴氰菊酯诱导小菜蛾成虫蛋白质组表达变化, 这为进一步筛选抗性靶标提供很大的帮助。     

蛋白质组中蛋白质磷酸化研究进展

随着后基因组时代的到来 ,对生命体器官、组织或细胞的全部蛋白质的表达、修饰及相互作用的研究已成为蛋白质组学的重要任务。蛋白质磷酸化是细胞内信号转导和酶调控最常见的机制之一 ,人类基因组约 2 %的基因编码 5 0 0种激酶和 10 0种磷酸酶。蛋白质磷酸化和去磷酸化作为原核和真核细胞表达调控的关键环节 ,了解其对功能的影响可以深入理解生命系统在分子水平的调控状况。目前蛋白质组磷酸化研究仍是功能基因组面临的重大课题 ,本文对此作一综述     

蛋白质组氨酸磷酸化研究进展

摘要：蛋白质磷酸化是一种可逆的翻译后修饰,这种翻译后修饰可以改变蛋白质的构象,进而使蛋白质活化或者失活。组氨酸磷酸化在细胞信号传导过程中发挥着重要作用,且组氨酸磷酸化与人类某些疾病密切相关,然而,由于组氨酸磷酸化含有P-N键,具备不稳定性,有关于组氨酸磷酸化的报道远远少于其它磷酸化的报道。本综述系统的总结了组氨酸磷酸化在生物学过程中的作用,以及近些年取得的重要研究进展,以期对深入研究组氨酸磷酸化提供理论参考。     

印楝素乳油对小菜蛾种群的控制作用模拟

应用害虫种群系统控制控制的状态空间方程和作用因子添加分析法,模拟1％印楝素乳油对小菜蛾种群的控制作用,结果表明,印楝素浮油对小菜蛾种群的控制机理,主要是对小菜蛾成虫的显忌避作用,尤其是在种群发展的初期,由于其显的忌避作用,降低田间卵量,使其种群数量明显降低下来,有效控制其为害。     

Behavioral and physiological responses of susceptible and resistant diamondback moth larvae to Bacillus thuringiensis

To determine whether field-selected resistance of diamondback moth (Plutella xylostella L.) (Lepidoptera: Plutellidae) to Bacillus thuringiensis is based on behavioral or physiological adaptation, we measured mortality, consumption, and movement of larvae from a susceptible and a resistant colony when placed on untreated and B. thuringiensis treated cabbage. Colonies did not differ in mortality, consumption, or movement on untreated cabbage. However, for a given amount of consumption of treated cabbage, resistant larvae had lower mortality than susceptible larvae, demonstrating that resistance had a physiological basis. The movement patterns could not account for the differences between colonies in survival. Resistant larvae did not avoid B. thuringiensis more than did susceptible larvae. Thus, we found no evidence for behavioral resistance.     

Age-dependent response to insecticides and enzymatic variation in susceptible and resistant codling moth larvae

Insecticide resistance in the codling moth, Cydia pomonella, partly results from increased metabolic detoxification. The aim of this study was to follow the age variations in larval susceptibility to deltamethrin and teflubenzuron in one susceptible (S) strain, and two resistant (Rv and Rt) ones selected for resistance to deltamethrin and diflubenzuron, respectively. The age variation of the activities of cytochrome P450-dependent monooxygenase (MFO), glutathione S-transferases (GST), and esterases in S and both resistant strains were simultaneously investigated. The highest levels of insecticide resistance were recorded in late instars in both resistant strains, although Rv neonates exhibited enhanced resistance to deltamethrin. The involvement of an additional deltamethrin-specific mechanism of resistance, which could be mainly expressed in early instars, was supported by previous demonstration of a kdr point mutation in the Rv strain. The cross-resistance between deltamethrin and teflubenzuron indicated the involvement of non-specific metabolic pathways in resistance to teflubenzuron, rather than target site modification. A positive correlation between enhanced GST activities and deltamethrin resistance suggested that this mechanism might take place into the adaptive response of C. pomonella to pyrethroids treatments. Enhanced MFO activity was recorded in each instar of the two resistant strains compared to the susceptible one. But these activities were not correlated to the responses to deltamethrin nor to teflubenzuron. In the light of these findings, studying age-dependence of responses to selection is central to the implementation of monitoring tests of resistances, especially if the target instars are difficult to collect in the field.     

Phosphorylation of ankyrin down-regulates its cooperative interaction with spectrin and protein 3

Ankyrin mediates the primary attachment between beta spectrin and protein 3. Ankyrin and spectrin interact in a positively cooperative fashion such that ankyrin binding increases the extent of spectrin tetramer and oligomer formation (Giorgi and Morrow: submitted, 1988). This cooperative interaction is enhanced by the cytoplasmic domain of protein 3, which is prepared as a 45-41-kDa fragment generated by chymotryptic digestion of erythrocyte membranes. Using sensitive isotope-ratio methods and nondenaturing PAGE, we now demonstrate directly (1) the enhanced affinity of ankyrin for spectrin oligomers compared to spectrin dimers; (2) a selective stimulation of the affinity of ankyrin for spectrin oligomer by the 43-kDa cytoplasmic domain of protein 3; and (3) a selective reduction in the affinity of ankyrin for spectrin tetramer and oligomer after its phosphorylation by the erythrocyte cAMP-independent membrane kinase. The phosphorylation of ankyrin does not affect its binding to spectrin dimer. Ankyrin also enhances the rate of interconversion between dimer-tetramer-oligomer by 2-3-fold at 30 degrees C, and in the presence of the 43-kDa fragment, ankyrin stimulates the rate of oligomer interconversions by nearly 40-fold at this temperature. These results demonstrate a long-range cooperative interaction between an integral membrane protein and the peripheral cytoskeleton and indicate that this linkage may be regulated by covalent protein phosphorylation. Such interactions may be of general importance in nonerythroid cells.     

Molecular switches for pheromone release from a moth pheromone-binding protein

Pheromone-binding proteins (PBPs) are involved in the uptake of pheromones from pores on the antennae, transport through an aqueous environment surrounding the olfactory receptor neurons, and fast delivery to pheromone receptors. We tested the hypothesis that a C-terminal segment and a flexible loop are involved in the release of pheromones to membrane-bound receptors. We expressed in Escherichia coli 11 mutants of the PBP from the silkworm moth, BmorPBP, taking into consideration structural differences between the forms with high and low binding affinity. The N-terminus was truncated and His-69, His-70 and His-95 at the base of a flexible loop, and a cluster of acidic residues at the C-terminus were mutated. Binding assays and circular dichroism analyses support a mechanism involving protonation of acidic residues Asp-132 and Glu-141 at the C-terminus and histidines, His-70 and His-95, in the base of a loop covering the binding pocket. The former leads to the formation of a new α-helix, which competes with pheromone for the binding pocket, whereas positive charge repulsion of the histidines opens the opposite side of the binding pocket.     

Oviposition of resistant and susceptible strains of Drosophila melanogaster in the presence of deltamethrin

Oviposition of three strains of Drosophila melanogaster in the presence of deltamethrin was observed. These strains had different levels of physiological susceptibility to deltamethrin. Two-choice tests were conducted with couples of flies in petri-dish arenas containing two oviposition dishes. On the first day of the experiment, females were given a choice between a treated oviposition dish and an untreated control dish. On the second day of the experiment, two control oviposition dishes were given to females. Although individual females showed a tendency to aggregate their eggs in one of the dishes, control experiments demonstrated an overall equal distribution of eggs between the dishes. When one of the two oviposition dishes in the arena was treated with deltamethrin, the percentage of females ovipositing and the mean number of eggs laid by females were reduced, compared with control arenas. Females avoided the treated oviposition dish and laid significantly more eggs on the control dish. Furthermore, when the deltamethrin concentration was increased on the first day, female flies postponed their oviposition and laid significantly more eggs on the second day. The resistant strain, SR, demonstrated the same capacity to select the untreated site for oviposition as the susceptible strain, but it showed a smaller oviposition reduction and egg retention. The relationship between physiological and behavioural susceptibility to deltamethrin is discussed.     

桃蚜抗吡虫啉品系和敏感品系某些生物学特性比较

室内连续筛选15代,桃蚜Myzuspersicae(Sulzer)对吡虫啉产生了一定水平的抗性,抗性倍数是14.34倍。通过建立抗性品系和敏感品系的生命表,比较了抗性品系和敏感品系的发育历期和生殖力的差异。结果表明,抗性品系适合度显著下降,若蚜存活率降低,若蚜历期延长,成蚜寿命缩短;在繁殖上表现为产仔量下降,生殖期缩短。以净生殖率(Ro)和内禀增长率(rm)来评价抗性品系的相对生物适合度,与敏感品系相比,吡虫啉抗性品系的相对适合度分别为0.699和0.897。     

Characterization and comparison of midgut proteases of Bacillus thuringiensis susceptible and resistant diamondback moth (Plutellidae: Lepidoptera)

The midgut proteases of the Bacillus thuringiensis resistant and susceptible populations of the diamondback moth, Plutella xylostella L. were characterized by using protease specific substrates and inhibitors. The midgut contained trypsin-like proteases of molecular weights of 97, 32, 29.5, 27.5, and 25 kDa. Of these five proteases, 29.5 kDa trypsin-like protease was the most predominant in activation of protoxins of Cry1Aa and Cry1Ab. The activation of Cry1Ab protoxin by midgut protease was fast (T(1/2) of 23-24 min) even at a protoxin:protease ratio of 250:1. The protoxin activation appeared to be multi-step process, and at least seven intermediates were observed before formation of a stable toxin of about 57.4 kDa from protoxin of about 133 kDa. Activation of Cry1Aa was faster than that of Cry1Ab on incubation of protoxins with midgut proteases and bovine trypsin. The protoxin and toxin forms of Cry proteins did not differ in toxicity towards larvae of P. xylostella. The differences in susceptibility of two populations to B. thuringiensis Cry1Ab were not due to midgut proteolytic activity. Further, the proteolytic patterns of Cry1A protoxins were similar in the resistant as well as susceptible populations of P. xylostella.     

Effect of an ecdysteroid agonist, tebufenozide, on the completion of diapause in susceptible and resistant strains of the codling moth, Cydia pomonella

Codling moth Cydia pomonella (L.) (Lepidoptera, Tortricidae) larvae kept under a short photoperiod enter a diapause which can be broken by exposure to 25°C following an extended chilling period. We investigated the effects of the ecdysteroid agonist tebufenozide on the diapause of this species, both on a laboratory strain susceptible to insecticides and two strains selected for resistance to diflubenzuron or deltamethrin. The treatment induced moulting by breaking diapause of the susceptible strain when applied at the end of the growth period, or by reducing the pre-emergence period when applied after chilling. The strains exhibiting resistance in the neonate instar were also resistant to the ecdysteroid agonist applied to diapausing larvae, indicating that mechanisms of resistance remained active during this arrest of development. High doses of tebufenozide were lethal for the most resistant insects, but neither reduced the pupation delay nor produced the larval-pupal moults which occur in the susceptible strain. As diapausing larvae of C. pomonella can be easily collected, the expression of resistance to tebufenozide both in the target instar, i.e. neonates, and during diapause may contribute to early detection of resistance to this insecticide in field populations.