奉节脐橙果实苯丙氨酸解氨酶活性及其基因表达与果皮褐变的关系

柑橘果皮褐变严重影响果实的商品价值和耐贮性．以奉节脐橙（Citrus sinensis Osbcck）果实为材料,通过采后常温、涂蜡、低温、机械损伤等处理研究了果实果皮褐变率、苯丙氨酸解氨酶（PAL）活性及PAL上基因在果皮褐变过程中表达水平的变化。结果表明,涂蜡、损伤处理均极显著地提高果实的果皮褐变率,而低温贮藏可显著降低其发生率;贮藏期问各处理的PAL活性均呈上升趋势,损伤处理PAL活性显著高于对照。果实发生褐变或受到机械损伤后,PAL2、PAL6基因的表达均比对照明显增强。结果首次表明脐橙果实PAL活性变化以及PAL2基因的表达与果皮褐变具有非常密切的关系．     

Increasing yeast secretion of heterologous proteins by regulating expression rates and post-secretory loss

Heterologous protein secretion involves the coupled processes of protein synthesis, protein folding, and secretory trafficking. A more complete understanding of how these processes interrelate could help direct optimization of secretion systems. Here we provide a detailed study regarding the dynamics of heterologous protein secretion from yeast in terms of intracellular protein levels, secreted protein levels, and unfolded protein response (UPR). Three different protein expression induction temperatures (20, 30, and 37 degrees C) were investigated as a means to modulate expression rates and thus cellular responses. Inducing at 20 degrees C yielded the slowest initial secretion rate, but the highest absolute level of product. Correspondingly, the level and the rate of both intracellular protein accumulation and unfolded protein response (UPR) activation were also the lowest at 20 degrees C. In addition, secretion ceased after approximately 22 h at 30 and 37 degrees C, respectively, while it was continuous until nutrient depletion at 20 degrees C. Maxima in secretion levels were observed that were a result of the additive effects of secretion cessation and post-secretory protein loss. The post-secretory loss of protein did not appear to result from solution phase proteolysis or aggregation, but required the presence of yeast cells. Refeeding of both yeast nitrogen base and casamino acids successfully prevented the post-secretory loss of protein at both high (37 degrees C) and low (20 degrees C) temperatures, and further increased secretion levels 1.5-fold at 20 degrees C where the secretory pathway was still functioning. Taken together, these findings suggest that there exists an appropriate balance between protein synthesis, processing and secretion rates required for secretion optimization.     

Production of phenylalanine ammonia-lyase (PAL): isolation and evaluation of yeast strains suitable for commercial production of l-phenylalanine

Summary Phenylalanine Ammonia-Lyase (PAL) containing microorganisms were isolated from a wide variety of natural habitats. The best 21 strains to emerge from the primary screen were screened for PAL activities in both directions using l-phenylalanine and t-cinnamate substrates. Twelve of the latter strains were compared for total cell production and PAL activity and 7 isolates were chosen for examination of the extent of PAL induction in various media. On the basis of these screens, isolate SPA 10 (identified as Rhodotorula rubra) was selected for further optimization. Growth was optimal at 28° C and pH 5.0, although cellular PAL activity was shown to be higher at sub-optimal temperatures (36° C) and pH (8.0) for growth. Synthesis of PAL was repressed when grown in the presence of various sugars and NH ₄ ⁺ ions. Manipulation of fermentation conditions enabled PAL synthesis to occur at maximum biomass levels, upon glucose exhaustion. PAL was rapidly inactivated within cells shortly after maximum synthesis was attained: feeding of d,l-isoleucine and low concentrations of d,l-phenylalanine, and shifting of fermentation temperature conferred catalyst stability for fermentations over 100 h. These results demonstrate the suitability and superiority of isolate SPA 10 for the commercial production of l-phenylalanine from trans-cinnamic acid.     

干旱胁迫对紫花苜蓿黄酮类化合物含量及其合成途径关键酶活性的影响

以3个紫花苜蓿(Medicago sativa)品种为试验材料,利用不同浓度(0、5%、10%、15％、20%、25%)的PEG 6000溶液模拟干旱胁迫,探究干旱胁迫对不同品种紫花苜蓿黄酮类化合物合成上游3个关键酶活性以及黄酮类化合物含量的影响。结果表明：(1)随着PEG 6000胁迫浓度的增加,3种紫花苜蓿叶片中PAL(苯丙氨酸解氨酶)、C4H(肉桂酸 4 羟基化酶)和4CL(4 香豆酸辅酶A连接酶)活性均呈先升高后降低的趋势,但不同酶类之间响应存在差异,即PAL活性在10% PEG 6000胁迫浓度下最高,C4H和4CL活性则在15% PEG 6000胁迫浓度下最高,且均显著高于相应对照。(2)3种紫花苜蓿植株地上部总黄酮含量和8种黄酮类化合物含量均随着PEG 6000胁迫浓度的增加呈先上升后下降的趋势,且均在10%~15% PEG 6000胁迫浓度时达到最高值,并显著高于相应对照。(3)各品种紫花苜蓿叶片黄酮类化合物含量与其关键酶活性呈不同程度的相关性,即紫花苜蓿黄酮类化合物合成途径上游3个关键酶活性与其地上部黄酮类化合物含量存在密切的关系。研究认为,不同程度的干旱胁迫可以通过促进黄酮类化合物合成途径上游关键酶活性的变化来调节紫花苜蓿植株中黄酮类化合物的合成,且适度干旱胁迫能显著促进相关酶活性增强和黄酮类化合物含量增加。     

两种氮素形态对药用植物三叶青中黄酮类活性成分积累的影响及相关分子机制的初步探讨

以珍稀濒危药用植物三叶青（Tetrastigma hemsleyanum Diels et Gilg）为材料,通过遮荫条件下（30%自然光）的盆栽试验,研究了硝态氮（NO₃^--N）和铵态氮（NH₄⁺-N）两种不同氮素形态对其根和叶中黄酮类活性成分含量的影响并探讨其机制。结果表明：NO₃^--N处理后的三叶青叶中原花青素B1、芦丁、山柰酚-3-O-芸香糖苷、槲皮素、异槲皮苷、山柰酚的含量比NH₄⁺-N处理的高;其根中的原花青素B1、芦丁、山柰酚-3-O-芸香糖苷、槲皮素、异槲皮苷、山柰酚的含量比NH₄⁺-N处理的高。进一步的研究发现NO₃^--N处理过的三叶青其根、叶中与黄酮类化合物合成相关的酶苯丙氨酸解氨酶（PAL）、查尔酮合成酶（CHS）转录与表达水平高于NH₄⁺-N处理的三叶青。此外,NO₃^--N处理的三叶青叶、根中的PAL和CHS酶活性也高于NH₄⁺-N处理。利用PAL特异性抑制剂AOA处理后,AOA能显著抑制PAL酶活性（P<0.05）并导致黄酮类成分含量极显著下降（P<0.01）。综上所述,施用NO₃^--N更有益于三叶青黄酮类化合物的生物合成,其主要是通过增加PAL代谢途径关键酶的基因表达量进而提高其酶活性来增强黄酮类化合物的合成。     

Wound-induced PAL activity is suppressed by heat-shock treatments that induce the synthesis of heat-shock proteins

Wounding lettuce leaves induces the de novo synthesis of phenylalanine ammonia-lyase (PAL, EC 4.3.1.5), the accumulation of phenolic compounds, and subsequent tissue browning. A brief heat-shock at 45°C reduces the rise in wound-induced PAL, the accumulation of phenolic compounds, and tissue browning. The activity of PAL measured 24 h after wounding and the content of phenolic compounds (absorbance of methanol extract at 320 nm) measured 48 h after wounding was highly correlated (R² > 0.90) in tissue developing the normal wound response and in tissue subjected to 0–180 s of heat-shock after wounding. The synthesis of a unique set of proteins called heat-shock proteins (hsps) is induced by these heat-shock treatments. Western-blot analyses of proteins isolated from wounded and heat-shocked Iceberg and Romaine lettuce mid-rib leaf tissue was done using antibodies against hsp 23. Only those heat-shock treatments that were effective at inducing the synthesis of hsp 23 were effective in reducing the activity of PAL induced by wounding and the subsequent accumulation of phenolic compounds. Hsps induced in non-wounded, whole leaves by exposure to 45°C for 150 s did not significantly interact with PAL previously synthesized in non-heat-shocked wounded leaves to limit its activity. The preferential synthesis of hsps over that of wound-induced PAL, rather than the presence of hsps, may be responsible for the ability of a heat-shock treatment to reduce the wound-induced increase in PAL activity. Our results support this novel concept, and the possibility that heat-shock treatments can have significant physiological effects on the response of the tissue to other stresses, not because of the specific genes they induce or repress, or the products they cause to be synthesized, but by their secondary action of influencing the synthesis of other proteins (e.g. PAL) by the suppression of non-hsps protein synthesis.     

增强UV-B辐射和氮对谷子叶光合色素及非酶促保护物质的影响

以谷子(Setaria italica(L)Beauv.)为对象,从拔节期开始持续给予低氮(1.875 mmol/L)和高氮(15 mmol/L)两种氮供应条件并从抽穗期开始进行26 d两种强度(4.29、7.12 kJ·m-2·d-1)的增强UV-B辐射处理,研究了谷子叶中光合色素含量、类黄酮含量和苯丙氨酸解氨酶(PAL)活性的变化.结果表明:与高氮供应条件相比,低氮供应条件明显降低了谷子叶中光合色素含量但提高了类胡萝卜素/叶绿素含量比值;在开花期中段和灌浆期中段,高氮供应条件下谷子叶中光合色素含量对增强UV-B辐射比低氮供应条件下的谷子更敏感.从灌浆期开始到处理结束,两种影响因子对谷子叶中类黄酮含量均有显著影响,增强UV-B辐射导致谷子叶中类黄酮含量逐渐升高,且相同增强UV-B辐射强度下低氮供应条件下的谷子叶中类黄酮含量明显高于高氮供应条件下的谷子.谷子叶中PAL活性对两种影响因子的响应较类黄酮含量更加敏感,低氮供应条件使谷子叶中PAL活性明显提高.结合上述指标的相关性分析结果可知,低氮供应条件加强了处于繁殖期主要阶段的谷子叶中类黄酮的积累,并使谷子叶中的类胡萝卜素/叶绿素含量比值明显提高,进而有助于维持谷子叶中光合色素含量在增强UV-B辐射条件下的相对稳定性,对植株抵抗UV-B辐射伤害有利.     

Influence of the environmental temperature on the metabolic use of dietary energy and nitrogen in the growing rat

Sixty-seven male rats of the Wistar CF strain were used in two trials to study the effect of ambient temperature on nitrogen and energy balances (experiment 1) and on the variations of certain biochemical parameters of metabolism (experiment 2). In both cases, the rats, housed individually in metal cages and fed ad libitum, were kept under three different temperatures : 21 degrees C (control), 3 degrees C (cold), 31 degrees C (hot). Compared to the controls, the rats at 3 degrees C showed a significant increase (P less than 0.01) in food intake and a significant decrease (P less than 0.01) in daily growth rate and feed efficiency ratio; body fat content was unchanged. Compared to the controls, the rats at 31 degrees C showed a decrease (P less than 0.01) in food intake and growth rate, a better feed efficiency ratio (P less than 0.01) and a significant increase in carcass lipid content. When compared to rats at 21 degrees C, those at 3 degrees C had a much lower nitrogen retention; at 31 degrees C this retention was only slightly lower. The urinary nitrogen excretion, increased by the cold temperature, reflected higher nitrogen catabolism, while its increase at a high temperature was probably due to a decrease in protein synthesis, and a subsequent degradation of excess ingested nitrogen. Energy retention increased (P less than 0.01) as the ambient temperature increased from 3 to 31 degrees C, and wide variations in the amounts of fixed energy in the form of proteins or lipids were observed. The balance results obtained have been discussed in relation to the variations of some biochemical parameters of metabolism such as free fatty acids, free glycerol, lipoprotein-lipase activity and in vitro utilization of labelled glucose.