高产谷胱甘肽酵母菌株的筛选和抗乙硫氨酸突变株的选育

[目的]选育高产谷胱甘肽的酵母菌株。[方法]通过紫外线进行诱变处理,运用推理育种技术获得抗乙硫氨酸突变株。[结果]从土壤中筛选出一株高产GSH的菌株HSJB1,其摇瓶发酵生物量为3.87 g/L,GSH产量为91.87 mg/L。依据菌株的细胞形态特征及生理生化特征,初步鉴定其为Saccharomyces cerevisiae。通过紫外线对出发菌株HSJB1进行诱变处理,获得一株抗乙硫氨酸突变株YBS77,该突变株经摇瓶发酵,生物量达7.60 g/L,GSH产量为211.96 mg/L。[结论]通过选育获得的突变株生物量比出发菌株提高了96.38%,GSH产量提高了130.72%,表明该育种方法可行。     

Effect of glutathione on gelatinization and retrogradation of wheat flour and starch

Reduced glutathione (GSH) commonly exists in wheat flour and has remarkable influence on gluten properties. In this study, effect of GSH on the gelatinization and retrogradation of wheat flour and wheat starch were investigated to better understand the GSH-gluten-starch interactions in wheat flour. Compared with wheat starch, wheat flour showed significant decreases in peak and final viscosity, and gelatinization onset temperature with increasing GSH concentration. GSH depolymerized gluten and thereby broke down the protein barrier around starch granules to make the starch easily gelatinized. However, the interaction between GSH and wheat starch restrained starch swelling. GSH addition resulted in weakened structure with higher water mobility in freshly gelatinized wheat flour dispersions but decreased water mobility in wheat starch dispersions. After storage at 4 °C for 7 d, GSH increased elasticity and retrogradation degree in wheat flour dispersions but retarded retrogradation in wheat starch dispersions. The results indicated that GSH promoted retrogradation of wheat flour, which mainly attributed to the depolymerized gluten embedding in the leached starch chains, and inhibiting the re-association of amylose, and subsequently promoted the starch intermolecular associations and starch retrogradation. This study could provide valuable information for the control of the quality of wheat flour-based products.     

Possible Relationship Between Performance and Oxidative Stress in Endurance Horses

The purpose of this study was to evaluate oxidative stress, antioxidant biomarkers, and performance during a multiday 210-km endurance race. Nine endurance athlete horses participated in this study. Samples were always taken at the same times of day, before the beginning of the race and after every day of competition. Analytic measurements included glutathione reductase (GR) and catalase activity, thiobarbituric acid–reactive substances (TBARs), and reactive carbonylated derivatives. Competition intensity was low, with an average speed of 12.56 ± 0.9 km/h. Four horses were unable to finish the race because of metabolic problems or fatigue. GR activity increased progressively (P < .001) throughout the competition, and TBARs showed a significant rise compared with baseline values (P < .01) but remained at the same levels throughout the 3 days of competition. Catalase and reactive carbonylated derivatives did not show any significant alterations in any time period. The best performance was obtained from horses who demonstrated higher GR capacity and/or lower TBAR concentration. In conclusion, redox status seems to modulate horses' performance in endurance races, but further studies are needed to better determine the adequate oxidant/antioxidant ratio to acquire optimal performance.     

奥利亚罗非鱼和尼罗罗非鱼几种谷胱甘肽S-转移酶基因克隆与分析

通过RT-PCR法和RACE法,分别从奥利亚罗非鱼(Oreochromisco aureus)肝脏获得alpha、rho1、rho2型GST(GSTA、GSTR1、GSTR2)基因cDNA全序列,从尼罗罗非鱼(Oreochromis nilotica)肝脏获得GSTR1、GSTR2基因cDNA全序列。结果表明:奥利亚罗非鱼GSTA基因cDNA全序列为933bp,5′非翻译区(5′-UTR)为98bp,3′非翻译区(3′-UTR)为166bp,开放阅读框(ORF)为669bp,编码222个氨基酸。奥利亚、尼罗罗非鱼GSTR1基因ORF均为681bp、均编码226个氨基酸;奥利亚、尼罗罗非鱼GSTR2基因ORF均为693bp,均编码230个氨基酸。氨基酸序列同源性比较和系统进化分析均表明,罗非鱼GST基因与鱼类GST同源性较高,与哺乳类、鸟类、两栖类GST同源性较低。     

MBF2转录调控小菜蛾谷胱甘肽S-转移酶代谢氯虫苯甲酰胺的功能

【目的】探究转录调控因子MBF2 (multiprotein bridging factor 2)调控小菜蛾（Plutella xylostella）体内谷胱甘肽S-转移酶（glutathione S-transferase,GST）的功能,及其在小菜蛾抗氯虫苯甲酰胺中的作用,为阐明小菜蛾对氯虫苯甲酰胺的解毒代谢机制提供理论依据。【方法】采用浸叶法测定敏感（SS）、广东连州（LZ）、云南通海（TH）3个小菜蛾种群对氯虫苯甲酰胺的抗性水平,酶动力学法测定3个种群的GST活性;使用实时荧光定量PCR(RT-qPCR)分析MBF2在不同抗性种群小菜蛾中的表达差异;采用氯虫苯甲酰胺LC50诱导12 h,分析其对小菜蛾MBF2的诱导表达作用;应用RNA干扰（RNA interference,RNAi）技术研究MBF2对GST基因的调控作用,并测定沉默MBF2后小菜蛾的GST活性;结合沉默后小菜蛾对药剂的敏感性变化,验证MBF2在小菜蛾抗氯虫苯甲酰胺中的作用。【结果】两个抗性种群（TH和LZ）相较SS种群分别达到了中抗及高抗的水平,TH和LZ抗性倍数分别为54.27和289.58;TH和LZ小菜蛾...     

Effect of diet and β-naphthoflavone on hepatic and renal glutathione S-transferase isoenzymes in carp (Cyprinus carpio)

Glutathione S-transferase (GST) isoenzymes were isolated from liver and kidney of carp (Cyprinus carpio) by glutathione affinity chromatography and chromatofocusing. Ten hepatic and eight renal catalytically active isoenzymes were identified. GST subunits from purified isoenzymes were further separated by high-performance liquid chromatography (HPLC) and were used as standards for the experimental sample analysis. Experimental samples came from carp that were fed fish meal (standard diet) or soybean bas ed diets for one year, injected or not with -naphthoflavone (BNF; i.p. injection of 50 mg kg-1). HPLC did not allow us to identify precisely the GST isoenzyme pattern in experimental carp. However, GSTs could be pooled in three categories: homodimeric, heterodimeric and unidentified GST subunits. On this basis, the effect of diet and BNF on the GST isoenzyme pattern was investigated. The homodimer/heterodimer ratio was decreased in liver of carp fed a standard diet and in kidney of both dietary groups. BNF increased the total specific GST activity in liver and kidney. However, the GST isoenzyme pattern was not modified in carp fed the standard diet while tissue specific modifications occured in carp fed the soybean diet. BNF decreased the homodimer/heterodimer ratio in liver and increased it in kidney. Abbreviations: BNF - -naphthoflavone; CDNB - 1-chloro-2,4-dinitrobenzene; GSH - reduced glutathione; GST - glutathione S-transferase; HPLC - high-performance liquid chromatography; SDS-PAGE - SDS-polyacrylamide gel electrophoresis.     

Supra-nutritional dietary intake of selenite and selenium yeast in normal and stressed rainbow trout (Oncorhynchus mykiss): Implications on selenium status and health responses

Commercial salmonid diets are not typically supplemented with selenium (Se) as they naturally contain relatively high levels of this essential trace element; however supplementation may be necessary to meet requirements during physical stress. Adopting an integrated approach by simultaneously determining Se status, various health parameters and interactions with other trace elements, this study aimed to determine the role of supra-supplemented selenite and Se-yeast (Selplex^®) on the growth and health of rainbow trout (Oncorhynchus mykiss), both under normal conditions and after exposure to chronic physical stress. Fish were fed one of seven diets; a basal un-supplemented diet (0.73 mg kg^− 1 Se) or diets supplemented with Se-yeast or sodium selenite to provide 2, 4, or 8 mg kg^− 1 Se for 10 weeks prior to subjection of daily handling and confinement stressors for seven days. Net whole body Se retention was significantly greater for Se-yeast than selenite at all levels. In normal conditions activities of hepatic glutathione peroxidase (GSH-Px) and thioredoxin reductase (Trx-R) indicate that Se requirements were met by the basal diet. Similarly, pre-stress supra-supplementation of selenite and Se-yeast did not affect oxidative status (total antioxidant capacity of serum and hepatic malondialdehyde), immuno-competence (respiratory burst activity, serum lysozyme, and leukocyte counts) or other haematological and growth parameters (nuclear abnormalities in erythrocytes, and specific growth rate). In contrast, the trend towards a higher GSH-Px post-stress in Se supplemented diets over the basal diet, particularly in Se-yeast fed fish, indicates that Se requirements may not be met by the un-supplemented practical diet in stressed fish. Seven days of chronic physical stress decreased whole body Se and increased GSH-Px activity signifying an increased Se utilisation. During stress Se status was more effectively maintained by Se-yeast than selenite. Increased hepatic lipid peroxidation in stressed fish fed 8 mg kg^− 1 selenite indicates a possible pro-oxidant effect of selenite. A positive interaction was observed between dietary selenite and whole body copper, but no such interaction was observed with Se-yeast. This study concludes that physical stressors can result in an elevated Se utilisation and consequently supplementation of commercial diets may be necessary such that Se reserves are available as a contingency for stress.     

胡杨谷胱甘肽过氧化物酶PeGPX基因的克隆及转化植株耐盐性分析

胡杨（Populus euphratica Oliv.）具有极强抗盐碱能力.本实验室前期胡杨微阵列芯片数据结果显示：盐胁迫下,胡杨谷胱甘肽过氧化物酶基因（PeGPX）的转录上调,暗示该基因可能对胡杨耐盐性具有一定的作用.为分析GPX对植物耐盐性的贡献,本研究以胡杨为材料,利用RT-PCR方法克隆了胡杨谷胱甘肽过氧化物酶PeGPX基因,并在烟草中过量表达该基因,以分析谷胱甘肽过氧化物酶活性与植物耐盐性的关系.研究结果显示,实验中克隆的cDNA （PeGPX）编码谷胱甘肽过氧化物酶,其ORF为693 bp,其蛋白由231个氨基酸编码.过量表达PeGPX基因的烟草与野生型烟草的耐盐性实验结果显示,野生型烟草植株在加NaCl （200 mmol/L）的MS培养基中生长15 d后,无明显的长高,且不长根;而转基因烟草在同样的加盐培养基上,生长基本没有受到抑制,植株生长状态良好,并且能够长根.光合数据显示,在盐胁迫下过量表达PeGPX基因烟草的净光合速率受到影响明显小于野生型烟草的净光合速率.酶活数据显示,转基因株系GPX酶活与野生型的相比在盐胁迫下活性有非常显著的提高.我们的研究结果说明：过表达PeGPX基因使得烟草的耐盐性得到显著提高,这对深入研究PeGPX基因在胡杨耐盐机制中的作用具有重要的意义.     

Non-steroidal inhibitors of Drosophila melanogaster steroidogenic glutathione S-transferase Noppera-bo

Insect growth regulators (IGRs) can be developed by elucidating the molecular mechanisms of insect-specific biological events. Because insect molting, and metamorphosis are controlled by ecdysteroids, their biosynthetic pathways can serve as targets for IGR development. The glutathione S-transferase Noppera-bo (Nobo), which is conserved in dipteran and lepidopteran species, plays an essential role in ecdysteroid biosynthesis. Our previous study using 17β-estradiol as a molecular probe revealed that Asp113 of Drosophila melanogaster Nobo (DmNobo) is essential for its biological function. However, to develop IGRs with a greater Nobo inhibitory activity than 17β-estradiol, further structural information is warranted. Here, we report five novel non-steroidal DmNobo inhibitors. Analysis of crystal structures of complexes revealed that DmNobo binds these inhibitors in an Asp113-independent manner. Among amino acid residues at the substrate-recognition site, conformation of conserved Phe39 was dynamically altered upon inhibitor binding. Therefore, these inhibitors can serve as seed compounds for IGR development.     

胁迫温度短期冲击对松毛虫赤眼蜂谷胱甘肽硫转移酶活性的影响

以松毛虫赤眼蜂为对象,对其不同温度条件下的谷胱甘肽硫转移酶比活力进行测定,结果表明:在15℃处理条件下,谷胱甘肽硫转移酶的比活力最低,为6.80±4.71;在35℃处理条件下最高,为98.22±1.76;25℃处理介于两者之间,为32.49±2.60;3种温度条件下的谷胱甘肽硫转移酶比活力存在显著差异(P0.05),且随着温度的升高比活力显著上升。