高温对家蚕三品系血淋巴中糖水平的影响（英文）

家蚕Bombyx mori的两个二化性品系热耐受型NB4D2和热敏感型CSR2均适合于温带气候,而多化性的PM（Pure Mysore) 品系适合于热带气候,将这3种品系5龄幼虫分别置于32℃和36℃的高温下,观察高温对其5龄幼虫至蛹期血淋巴中糖含量及海藻糖酶活性的影响。结果表明： PM幼虫和蛹的死亡率均小于NB4D2和CSR2。在蜕皮期间血淋巴海藻糖水平较高,而葡萄糖水平及海藻糖酶活性较低。32℃和36℃的高温下,幼虫蜕皮期间血淋巴中糖含量及海藻糖酶活性仅在其各自的水平上表现为小幅度的增加。蜕皮后幼虫血淋巴中海藻糖含量显著下降,而葡萄糖含量和海藻糖酶活性显著上升。在较高温度下,蜕皮后幼虫血淋巴中海藻糖含量下降幅度更大,而葡萄糖含量及海藻糖酶活性上升水平也更加显著。25±1℃下取食幼虫血淋巴中葡萄糖含量显著下降,海藻糖含量显著上升;3℃和36℃下PM 和NB4D2取食幼虫血淋巴葡萄糖和海藻糖含量以及海藻糖酶活性增加,而CSR2均减少或降低。吐丝幼虫血淋巴中葡萄糖含量及海藻糖酶活性显著下降,海藻糖小幅度下降。而在较高温度下,耐热型PM 和NB4D2吐丝家蚕血淋巴糖含量含量和海藻糖酶活性明显增加,而热敏感型CSR2的则明显下降。这3种品系蛹发育期的血淋巴糖含量及海藻糖酶活性均下降。在两较高温度下,PM蛹期血淋巴糖和海藻糖酶活性增加,而NB4D2 36℃时增加幅度小于32℃时。对于CSR2,32℃时观察到其血淋巴葡萄糖含量增加,但当环境温度增加到36℃时其血淋巴葡萄糖含量降至正常水平下。然而,当CSR2的蛹置于32℃和36℃时血淋巴海藻糖含量及其酶活性下降,且36℃时下降幅度更大。因此,桑蚕对高温的适应取决于家蚕的品系及发育阶段,并可通过其血淋巴糖及海藻糖酶活性水平进行验证。     

海藻糖降解酶抑制因子的研究

研究了一系列因素对食尼古丁节杆菌D-97胞内海藻糖降解酵的抑制作用,发现有效霉素(井冈霉素)和乙酸能有效抑制海藻糖降解酶的活性,抑制率最大分别达到87．13％和85．16％．同时研究了它们对海藻糖转化率的影响,发现有效霉素能提高海藻糖转化率,最大增幅达17．44％,而乙酸却降低了转化率。     

Effect of trehalose on protein structure

Trehalose is a ubiquitous molecule that occurs in lower and higher life forms but not in mammals. Till about 40 years ago, trehalose was visualized as a storage molecule, aiding the release of glucose for carrying out cellular functions. This perception has now changed dramatically. The role of trehalose has expanded, and this molecule has now been implicated in a variety of situations. Trehalose is synthesized as a stress‐responsive factor when cells are exposed to environmental stresses like heat, cold, oxidation, desiccation, and so forth. When unicellular organisms are exposed to stress, they adapt by synthesizing huge amounts of trehalose, which helps them in retaining cellular integrity. This is thought to occur by prevention of denaturation of proteins by trehalose, which would otherwise degrade under stress. This explanation may be rational, since recently, trehalose has been shown to slow down the rate of polyglutamine‐mediated protein aggregation and the resultant pathogenesis by stabilizing an aggregation‐prone model protein. In recent years, trehalose has also proved useful in the cryopreservation of sperm and stem cells and in the development of a highly reliable organ preservation solution. This review aims to highlight the changing perception of the role of trehalose over the last 10 years and to propose common mechanisms that may be involved in all the myriad ways in which trehalose stabilizes protein structures. These will take into account the structure of trehalose molecule and its interactions with its environment, and the explanations will focus on the role of trehalose in preventing protein denaturation.     

红色亚栖热菌TPS/TPP海藻糖合成途径中相关基因的克隆、表达及功能鉴定

通过构建红色亚栖热菌(Meiothermus ruberCBS-01)的基因组DNA文库,克隆得到该嗜热菌海藻糖合成途径中的磷酸海藻糖合成酶(TPS)和磷酸海藻糖磷酸酯酶(TPP)基因。以pET21a为表达载体,将磷酸海藻糖合成酶和磷酸海藻糖磷酸酯酶在大肠杆菌中进行表达并纯化,利用薄层层析的方法验证了这两个酶的活性。同时,本研究检测了红色亚栖热菌在各种环境压力下细胞内含物成分的变化情况,发现在高渗环境压力的诱导下,该菌会在胞内积累大量的6-磷酸海藻糖,而并非海藻糖,这为进一步研究TPS/TPP和TreS途径在细胞体内的作用奠定了基础。     

Different mechanisms of the biochemical adaptation of mycelial fungi to temperature stress: Changes in the cytosol carbohydrate composition

The effect of temperature stress on the cytosol carbohydrate composition of fungi belonging to various systematic groups was investigated. InMucorales representatives (subkingdomEomycota, phylumArchetnycota, classZygomycetes), adaptation to hypo- and hyperthermia occurs via the regulation of trehalose synthesis, although inositol is also involved in these processes inBlakeslea trispora. InAscomycota (subkingdomNeomycota), oversynthesis of mannitol and glycerol occurs under hypothermia, whereas oversynthesis of trehalose and inositol takes place under hyperthermia.Basidiomycota (subkingdomNeomycota) use two pathways of biochemical adaptation, depending on the cytosol carbohydrate composition. In the absence of sucrose, glycerol and arabitol are involved in the adaptation to hyperthermia; trehalose accumulates under hypothermic conditions (type I of regulation). Type II regulation (revealed inPleurotus ostreatus) involves sucrose rather than glycerol or arabitol. The data obtained are discussed in terms of fungal systematics and phylogeny.     

Genetic modification of photosynthesis with E. coli genes for trehalose synthesis

Improvement in photosynthesis per unit leaf area has been difficult to alter by breeding or genetic modification. We report large changes in photosynthesis in Nicotiana tabacum transformed with E. coli genes for the trehalose pathway. Significantly, photosynthetic capacity (CO2 assimilation at varying light and CO2, and quantum yield of PSII electron transport) per unit leaf area and per leaf dry weight were increased in lines of N. tabacum transformed with the E. coli gene otsA, which encodes trehalose phosphate synthase. In contrast, transformation with otsB, which encodes trehalose phosphate phosphatase or Trec, encoding trehalose phosphate hydrolase, produced the opposite effect. Changes in CO2 assimilation per unit leaf area were closely related to the amount and activity of Rubisco, but not to the maximum activities of other Calvin cycle enzymes. Alterations in photosynthesis were associated with trehalose 6-phosphate content rather than trehalose. When growth parameters were determined, a greater photosynthetic capacity did not translate into greater relative growth rate or biomass. This was because photosynthetic capacity was negatively related to leaf area and leaf area ratio. In contrast, relative growth rate and biomass were positively related to leaf area. These results demonstrate a novel means of modifying Rubisco content and photosynthesis, and the complexities of regulation of photosynthesis at the whole plant level, with potential benefits to biomass production through improved leaf area.     

Enzymatic processes for the purification of trehalose

A dual‐enzyme process aiming at facilitating the purification of trehalose from maltose is reported in this study. Enzymatic conversion of maltose to trehalose usually leads to the presence of significant amount of glucose, by‐product of the reaction, and unreacted maltose. To facilitate the separation of trehalose from glucose and unreacted maltose, sequential conversion of maltose to glucose and glucose to gluconic acid under the catalysis of glucoamylase and glucose oxidase, respectively, is studied. This study focuses on the hydrolysis of maltose with immobilized glucoamylase on Eupergit® C and CM Sepharose. CM Sepharose exhibited a higher protein adsorption capacity, 49.35 ± 1.43 mg/g, and was thus selected as carrier for the immobilization of glucoamylase. The optimal reaction temperature and reaction pH of the immobilized glucoamylase for maltose hydrolysis were identified as 40°C and 4.0, respectively. Under such conditions, the unreacted maltose in the product stream of trehalose synthase‐catalyzed reaction was completely converted to glucose within 35 min, without detectable trehalose degradation. The conversion of maltose to glucose could be maintained at 0.92 even after 80 cycles in repeated‐batch operations. It was also demonstrated that glucose thus generated could be readily oxidized into gluconic acid, which can be easily separated from trehalose. We thus believe the proposed process of maltose hydrolysis with immobilized glucoamylase, in conjunction with trehalose synthase‐catalyzed isomerization and glucose oxidase‐catalyzed oxidation, is promising for the production and purification of trehalose on industrial scales. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 2013     

Regulation of growth by the trehalose pathway: Relationship to temperature and sucrose

Carbon signaling can override carbon supply in the regulation of growth. At least some of this regulation is imparted by the sugar signal trehalose 6-phosphate (T6P) through the protein kinase, SnRK1. This signaling pathway regulates biosynthetic processes involved in growth under optimal growing conditions. Recently, using a seedling system we showed that under sub-optimal conditions, such as cold, carbon signaling by T6P/ SnRK1 enables recovery of growth following relief of the stress. The T6P/ SnRK1 mechanism thus could be selected as a means of improving low temperature tolerance. High-throughput automated Fv/Fm measurements provide a potential means to screen for T6P/ SnRK1, and here we confirm through measurements of Fv/Fm in rosettes that T6P promotes low temperature tolerance and recovery during cold to warm transfer. Further, to better understand the coordination between sugars, trehalose pathway, and temperature-dependent growth, we examine the interrelationship between sugars, trehalose phosphate synthase (TPS), and trehalose phosphate phosphatase (TPP) gene expression and T6P content in seedlings. Sucrose, particularly when fed exogenously, correlated well with TPS1 and TPPB gene expression, suggesting that these enzymes are involved in maintaining carbon flux through the pathway in relation to sucrose supply. However, when sucrose accumulated to higher levels under low temperature and low N, TPS1 and TPPB expression were less directly related to sucrose; other factors may also contribute to regulation of TPS1 and TPPB expression under these conditions. TPPA expression was not related to sucrose content and all genes were not well correlated with endogenous glucose. Our work has implications for understanding acclimation to sink-limited growth conditions such as low temperature and for screening cold-tolerant genotypes with altered T6P/ SnRK1 signaling.     

Feedforward Regulation of Glucose Metabolism by Steroid Hormones Drives a Developmental Transition in Drosophila

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