产1,3-丙二醇新型基因工程菌的构建

以甘油为底物转化生产1,3-丙二醇的生物合成途径中,往往由于还原力NADH的不足,限制了1,3-丙二醇的合成,引起中间代谢产物3-羟基丙醛累积,进而抑制甘油脱水酶的活性,阻碍菌体的生长,严重影响1,3-丙二醇的合成途径.为了解决合成途径中还原力不足这一主要矛盾,本文以大肠杆菌和克雷伯氏菌染色体DNA为模板克隆得到yqhD和dhaB、dhaT基因,构建双启动子表达载体pEtac-dhaB-tac-yqhD及表达载体pUC-tac-dhaT,成功共转入E.coli JM109,得到可利用两种辅酶(NADH、NADPH)将片油转化为1,3-丙二醇且传代稳定的重组大肠杆菌双质粒系统,发酵结果表明,1,3-丙二醇产量提高了28.6%.图6表1参17     

克雷伯氏菌产1,3-丙二醇菌株选育及甘油脱水酶基因转录分析

通过对野生克雷伯氏菌(Klebsiella)进行紫外诱变,获得一株耐高浓度甘油及高浓度1,3-丙二醇的突变菌株A-39-3,该突变株产1,3-丙二醇的量较野生菌株提高了58%,副产物2,3-丁二醇和乙醇的量分别降低了27%和19%.为探讨突变株高产1,3-丙二醇的原因,通过对突变菌株发酵过程中关键酶的酶活跟踪分析,发现1,3-丙二醇氧化还原酶(PDOR)的酶活力与野生菌株相差不大,甘油脱氢酶(GDH)的酶活力略有下降,而突变菌株的甘油脱水酶(GDHt)的酶活力明显高于野生菌株.并采用半定量RT-PCR方法,从转录水平上比较甘油脱水酶基因的差异,结果发现突变菌株A-39-3甘油脱水酶基因的相对mRNA转录水平是野生菌株K的2.58倍,分析说明该突变株1,3-丙二醇产量的提高与关键酶基因dhaB的mRNA转录水平关系密切.     

budC缺失对克雷伯氏菌联产3-羟基丙酸和1,3-丙二醇的影响

在利用克雷伯氏菌(Klebsiella pneumoniae)联产3-羟基丙酸和1,3-丙二醇过程中,副产物对碳流的竞争是限制产物合成的重要因素.通过将来源于大肠杆菌的醛脱氢酶基因在budC缺失型克雷伯氏菌中过表达,研究budC缺失对克雷伯氏菌联产1,3-丙二醇和3-羟基丙酸的影响.与K.pneumoniae ZG27/pUC19-aldH相比,缺失型菌株K.pneumoniae ZG40(budC?)/pUC19-aldH发酵60 h后,1,3-丙二醇和3-羟基丙酸的产量分别为64.0 mmol/L和134 mmol/L,3-羟基丙酸转化率提高了22%,副产物中除2,3-丁二醇外均有不同程度降低.本研究表明,过表达醛脱氢酶的重组克雷伯氏菌能够积累3-羟基丙酸,但在微氧发酵24 h后,1,3-丙二醇逐渐向3-羟基丙酸转化;而budC的缺失进一步促进了1,3-丙二醇向3-羟基丙酸的转化.     

促进氰钴胺素转化为腺苷钴胺素的基因表达与鉴定

辅酶B12作为甘油脱水酶(GDHt)的辅酶参与到Klebsiella pneumoniae代谢甘油生成1,3-丙二醇(1,3-PD)和3-羟基丙酸(3-HP)的途径中.前期研究表明底物甘油可以导致辅酶B12分子中Co—C键的断裂,进而引起GDHt的失活.为进一步研究非活性的维生素B12(CNCbI)转化为具有活性的辅酶B12(AdoCbI)的过程,从K.pneumoniae中克隆得到了ATP:钴(I)胺素腺苷转移酶(ACA)基因btuR和还原酶基因yciK,并成功构建了双启动子表达质粒pUC18-tac-btuR-tac-yciK,将表达质粒转化Escherichia coli JM109获得了一株重组菌.利用改进的分析方法,结果表明:1)重组蛋白具有腺苷转移酶的活性;2)重组菌可以很好地将非活性的钴胺素,如维生素B12,转化生成具有活性的辅酶B12.     

利用甘油脱水酶基因构建产3-羟基丙醛工程菌及其表达比较

3-羟基丙醛(3-HPA)是一种重要的化工产品,可由甘油经甘油脱水酶作用后生成.为获得产3-HPA基因工程菌,在已构建含甘油脱水酶基因及其激活因子大亚基质粒pEtac-dhaB-gdrA的基础上,构建了包含小亚基gdrB激活因子的重组质粒pEtac-dhaB-gdrA-gdrB.利用大肠杆菌通用tac启动子将该质粒在不同Escherichia coli BL21、DH5a及JM109中进行表达.阳性转化子经IPTG诱导后,提取总RNA,以cDNA为模板进行RT-PCR发现,目标基因在不同宿主都能较好转录.SDS-PAGE、酶活测定和3-HPA浓度测定结果表明,目标蛋白表达存在差异;酶活分别为4.7(±0.44)、3.5(±0.95)、8.1(±0.66)U/mg;发酵液中3-HPA的含量分别为0.012(±0.0044)、0.014(±0.003)、0.375(±0.018)g L-1,重组E.coli JM109/pEtac-dhaB-gdrA-gdrB具有较好的甘油脱水酶基因表达和产3-HPA性能.该基因工程菌与克雷伯氏菌(Klebsiella pneumoniae)相比,发酵副产物明显较少,有利于后期提取,为生产3-HPA提供了一条新思路.     

重组大肠杆菌产CotA漆酶的发酵条件优化

CotA漆酶在环境保护、食品工业和纸浆漂白等工业中具有重要的应用价值.将重组表达载体pET-22b/CotA转入大肠杆菌BL21(DE3),得到工程菌株,采用单因素实验和正交实验相结合的方法,研究诱导表达条件和发酵培养基对重组大肠杆菌产CotA漆酶量的影响.结果表明,初始pH 7.5的培养基中,添加0.6 mmol L-1 Cu2+,1 g L-1葡萄糖作碳源、15 g L-1蛋白胨和2 g L-1硫酸铵作氮源,以10%的接种量,37℃、200 r/min,直到菌液的D600 nm值为1.0,加入终浓度为1.0 mmol L-1的IPTG,25℃诱导12 h,漆酶的产量最高.优化前发酵液的粗提液的漆酶活性仅为1 190 U mL-1,优化后达到3 526 U mL-1,正交实验优化后漆酶活性提高了2.96倍.纯化的CotA漆酶最适反应温度为45℃,最适pH值为7.2.CotA漆酶对RBBR的脱色率在90%以上,此CotA漆酶在短时间内对染料能够有效地脱色,能够成为有潜力的工业用酶.图6表3参17     

顺丁烯二酸异构酶工程菌发酵条件优化

为提高重组大肠杆菌中顺丁烯二酸异构酶表达量,通过正交试验设计,对工程菌的生长条件和目的蛋白可溶表达条件进行优化.采用250 mL三角瓶中装有50 mL(Amp 100 mg L-1)的培养基,分别研究培养基中葡萄糖、蛋白胨、酵母浸粉的浓度,培养基pH值以及摇床转速、装液量、接种量等对蛋白可溶表达量的影响.确定顺丁烯二酸异构酶工程菌最优化培养基为:蛋白胨20 g L-1、酵母浸粉2.5 g L-1、K2HPO4·3H2O 3.0 g L-1、KH2PO4 1.5 g L-1、NaCl 6 g L-1、MgSO43 g L-1,培养基pH调至6.5.确定顺丁烯二酸异构酶工程菌可溶性表达最优条件为:37℃下培养至D600 nm值为1.0时,添加终浓度为0.05 mmol L-1的IPTG进行诱导,诱导温度37℃,摇床转速220 r min-1,装液量20%,接种量5%,诱导时长为6 h.利用BioFlo 415发酵罐以最优化的培养基和发酵条件对该工程菌进行了3批发酵实验,与摇瓶实验相比,顺丁烯二酸异构酶的表达量提高了近1.5倍,单位发酵液的酶活力由46 U mL-1发酵液提高到78 U mL-1发酵液.以上数据为顺丁烯二酸异构酶重组工程菌的中试发酵奠定了基础.图7表2参17     

嗜热脂肪芽孢杆菌过氧化氢酶基因perA在大肠杆菌中的高效表达

利用PCR技术得到嗜热脂肪芽孢杆菌 (Bacillusstearothermophilus)过氧化氢酶基因 perA ,将该基因与表达载体 pKK2 2 3 3连接构建重组质粒pK perA ,转化大肠杆菌过氧化氢酶HPⅠ和HPⅡ双缺突变株UM 2 ,得到重组大肠杆菌UM 2 1.酶活测定结果表明 ,表达产物具有正常的生物学活性 .SDS PAGE电泳结果显示出明显的特异性表达条带 ,单体Mr =86× 10 3 ,与嗜热脂肪芽孢杆菌所产酶相同 .实验表明 ,重组质粒在宿主UM 2中有较好的稳定性 ,在无选择压力条件下传代 6 0次基本保持稳定 ,传代 10 0次重组质粒保留 80 %以上 .摇瓶实验确定重组菌的最佳表达条件为 :IPTG浓度 ,0 .75mmol/L ;诱导时间 3h ;培养基起始 pH 6 .5 ;诱导温度 37℃ ;装液量 5 0mL/ 2 5 0mL .在优化条件下 ,重组菌产生的过氧化氢酶占菌体总蛋白的 8% ,酶活力可达 35U/mL ,是原始菌株BacillusstearothermophilusIAM110 0 1的 11.7倍 .图 2表 1参 10     

1株高效油脂降解菌株的筛选及其降解特性研究

从学校餐厅污泥中分离得到7株油脂降解菌。以芝麻油为唯一碳源,通过驯化培养、初筛和复筛得到1株芝麻油降解优势菌株。菌种特性研究表明其为好氧菌,降解芝麻油的最适温度为30~37℃左右,pH值为8.0;在最佳生长环境下,该菌株在含20g·L-1芝麻油的培养基中72h内对油脂的降解率达80%以上。     

嗜热子囊菌光孢变种β-葡萄糖苷酶的基因克隆、表达及酶学性质分析

从嗜热子囊菌光孢变种(Thermoascus aurantiacus var.levisporus)RNA中通过RT-PCR克隆出β-葡萄糖苷酶基因bgl Ⅰ的全长序列,cDNA序列为2 672 bp.Genbank登录号为EU269025,将该片段插入巴斯德毕赤酵母Pichia pastoris分泌型表达载体pPIC9K中,获得重组质粒pPIC9K/bgl,经线性化后用电穿孔法导入毕赤酵母GS115中,在醇氧化酶AOXI基因启动子作用下,获得高效表达β-葡萄糖苷酶的毕赤酵母工程菌株.经DEAE-Sepharose Fast Flow阴离子层析纯化了该重组表达蛋白.SDS-PAGER测得该重组蛋白相对分子质量(M)约为120×103.经甲醇诱导,培养基中β-葡萄糖苷酶的活力可达1.2 U/mg,小规模发酵量达0.45 mg/mL.该酶的最适反应温度为60℃,最适反应pH为5.0.于70℃保温30 min仍保持80%的酶活力,具有较高的热稳定性,在pH 3.0～9.0的条件下酸碱耐受性强.图6表1参22     

多溴联苯醚胁迫下鲫鱼肝脏微粒体CYP3A1和GST的响应

以鲫鱼Carassius auratus Linn.为试验鱼类,研究了其暴露于不同质量浓度的2,2',4,4'-四溴联苯醚(PBDE-47)和十溴联苯醚(PBDE-209)后鱼肝微粒体中CYP3A1和GST酶活性的动态变化.结果表明,鲫鱼在0.10～5.00 mg·L-1的PBDE-47和5.00～50.0 mg·L-1的PBDE-209中暴露15 d后,除0.10 mg·L-1PBDE-47、5.00 mg·L-1和10.0 mg·L-1PBDE-209试验组外,其余各试验组的鱼肝微粒体中CYP3A1被诱导,呈显著的剂量.效应关系.CYP3A1的活性随着作用时间的延续而上升,到试验第15天时达到最高,但上升速率最快的阶段为试验的第0-5天.而GST酶则表现出不同的特点,呈先升高后降低的趋势.经过15 d试验,除0.10 mg·L-1PBDE.47和5.00 mg·L-1PBDE-209以外的各试验组鲫鱼肝脏微粒体中的GST活性仅为对照组的12.74%～85.35%,表明PBDEs已对鱼体肝脏GST产生了较为严重的影响.研究表明,鱼类肝脏微粒体中CYP3A1和GST酶可作为污染生物标志物来评价PBDEs的早期污染毒理效应.     

螯台球菌TAD1在高温曝气生物滤池中的异养硝化-反硝化性能

初步实验证实螯台球菌(Chelatococcus daeguensis)TAD1在高温下具有异养硝化-反硝化的能力,为验证其可应用性,采用曝气生物滤池工艺,研究了TAD1在温度为50℃的异养硝化-反硝化性能.结果表明,TAD1在曝气生物滤池中可同时进行好氧反硝化和异养硝化.当分别以硝氮、氨氮及硝氮和氨氮为氮源时,12 h的氮去除率均达到100%,氮的去除能力分别为12.67 mg.L-.1h-1、3.62 mg.L-.1h-1及16.53 mg.L-.1h-1.虽然在脱氮过程中,亚硝盐在6 h迅速积累到76 mg.L-1(硝氮为氮源)和52.6 mg.L-1(硝氮和氨氮为氮源),但在随后的几个小时内又快速降低至0(检测限之外).因而,TAD1具有应用于高温生物脱氮工艺的能力和优势.     

Rabbit biocontrol and landscape‐scale recovery of threatened desert mammals

Funding for species conservation is insufficient to meet the current challenges facing global biodiversity, yet many programs use expensive single‐species recovery actions and neglect broader management that addresses threatening processes. Arid Australia has the world's worst modern mammalian extinction record, largely attributable to competition from introduced herbivores, particularly European rabbits (Oryctolagus cuniculus) and predation by feral cats (Felis catus) and foxes (Vulpes vulpes). The biological control agent rabbit hemorrhagic disease virus (RHDV) was introduced to Australia in 1995 and resulted in dramatic, widespread rabbit suppression. We compared the area of occupancy and extent of occurrence of 4 extant species of small mammals before and after RHDV outbreak, relative to rainfall, sampling effort, and rabbit and predator populations. Despite low rainfall during the first 14 years after RHDV, 2 native rodents listed by the International Union for Conservation of Nature (IUCN), the dusky hopping‐mouse (Notomys fuscus) and plains mouse (Pseudomys australis), increased their extent of occurrence by 241–365%. A threatened marsupial micropredator, the crest‐tailed mulgara (Dasycercus cristicauda), underwent a 70‐fold increase in extent of occurrence and a 20‐fold increase in area of occupancy. Both bottom‐up and top‐down trophic effects were attributed to RHDV, namely decreased competition for food resources and declines in rabbit‐dependent predators. Based on these sustained increases, these 3 previously threatened species now qualify for threat‐category downgrading on the IUCN Red List. These recoveries are on a scale rarely documented in mammals and give impetus to programs aimed at targeted use of RHDV in Australia, rather than simply employing top‐down threat‐based management of arid ecosystems. Conservation programs that take big‐picture approaches to addressing threatening processes over large spatial scales should be prioritized to maximize return from scarce conservation funding. Further, these should be coupled with long‐term ecological monitoring, a critical tool in detecting and understanding complex ecosystem change.     

芽孢杆菌发酵产碱性果胶酶温度控制策略

采用自行筛选获得一株产碱性果胶酶芽孢杆菌WSH03-09,在小型发酵罐中研究了不同温度对碱性果胶酶分批发酵的影响,结果表明,在恒定39℃条件下,可获得最高酶活5．39u／mL,各温度条件下的菌体干重相差不多,最终均能达11．5g／L左右;在发酵前期,控制温度41℃时最有利于菌体的生长,而在产物合成期,控制37℃有利于获得较高的产物合成比速,在此基础上,提出分阶段温度控制策略,采用此温度控制策略进行碱性果胶酶的发酵,碱性果胶酶酶活达5．99u／mL,比采用单一温度下的最大值提高了11％,其它各项指标也有较大提高．图6表1参6     

A rapid determination of histamine and 3-methylhistamine in human plasma by high performance liquid chromatography

Histamine plays an important pathophysiological role in allergy, inflammation, gastric acid secretion, microcirculation and neurotransmission. 3-Methylhistamine is a prominent metabolite of histamine. Different methods for determination of histamine in biological fluids have been developed. In the present study, a simple, simultaneous determination of histamine and 3-methylhistamine by HPLC (precolumn derivatization with fluorescamine) was developed in human plasma using fluorescence detection with 1-methylhistamine as the internal standard. Linear regression analysis of the ratios of the concentrations of histamine and 3-methylhistamine (X) against peak height ratios (Y) yielded the following: y = 0.0073x ? 0.0096 (R ² = 0.990) and y = 0.0077x ? 0.0111 (R ² = 0.989). In conclusion, it was possible to detect histamine and 3-methylhistamine below 5 ng mL^?1 in 1 mL plasma.     

1,3-丁二烯与DNA交联致癌机理的研究——代谢产物与鸟嘌呤反应及与DNA交联作用的AM1计算

采用AMI方法计算了环境致癌物1.2-环氧3,4-丁烯(EB)和1,2,3,4二环氧丁烷(DEB)与DNA鸟嘌呤反应过程速率控制步骤的活化能及DEB与DNA片段生成烷化交联产物的结构和能量、结果得出:用烷化反应的难易程度难以解释DEB的致突性比EB大100倍的实验事实;强致突的DEB可与鸟嘌吟发生两次烷化反应,生成DNA交联产物,交联后的DNA结构稳定、变形小:而EB则不能交联.这可能为两者基因毒性差异巨大的分子机制.     

利用环境一号卫星热红外通道反演太湖流域地表温度的3种方法比较

利用太湖流域2010年3月26日过境的1景环境一号卫星热红外数据(轨道号为451/77),根据同步modis数据反演大气水汽含量参数,分别利用覃志豪单窗算法、普适性单通道算法、基于影像的Artis反演算法反演太湖流域地表温度,通过与同期的MOD11_L2级modis温度产品进行对比分析,探寻适合于环境卫星热红外通道反演地表温度的方法,以达到对环境一号卫星热红外数据定量化应用示范的目的。结果表明,相比其他算法,普适性单通道算法精度较高,与modis温度产品温差较小,为+1.23 K,而环境一号卫星过境时间与modis温度产品时差为39 min,因此该方法的反演精度可以接受。     

菌株NKS-3对溴氨酸脱色特性探讨

从受溴氨酸污染地筛选出菌株ＮＫＳ－ ３ ,它对溴氨酸有脱色作用。菌株生长随碳源浓度增加而旺盛,溴氨酸能刺激菌株生长。溴氨酸浓度即使达到１０００ ｍｇ／Ｌ 也能被菌株基本脱色。研究发现,盐度抑制菌生长,在含盐培养液中溴氨酸的脱色过程被抑制     

Ability of Matrix Models to Explain the Past and Predict the Future of Plant Populations

Uncertainty associated with ecological forecasts has long been recognized, but forecast accuracy is rarely quantified. We evaluated how well data on 82 populations of 20 species of plants spanning 3 continents explained and predicted plant population dynamics. We parameterized stage‐based matrix models with demographic data from individually marked plants and determined how well these models forecast population sizes observed at least 5 years into the future. Simple demographic models forecasted population dynamics poorly; only 40% of observed population sizes fell within our forecasts’ 95% confidence limits. However, these models explained population dynamics during the years in which data were collected; observed changes in population size during the data‐collection period were strongly positively correlated with population growth rate. Thus, these models are at least a sound way to quantify population status. Poor forecasts were not associated with the number of individual plants or years of data. We tested whether vital rates were density dependent and found both positive and negative density dependence. However, density dependence was not associated with forecast error. Forecast error was significantly associated with environmental differences between the data collection and forecast periods. To forecast population fates, more detailed models, such as those that project how environments are likely to change and how these changes will affect population dynamics, may be needed. Such detailed models are not always feasible. Thus, it may be wiser to make risk‐averse decisions than to expect precise forecasts from models. Habilidad de los Modelos Matriciales para Explicar el Pasado y Predecir el Futuro de las Poblaciones de Plantas     

Meta-analysis of susceptibility of woody plants to loss of genetic diversity through habitat fragmentation

Shrubs and trees are assumed less likely to lose genetic variation in response to habitat fragmentation because they have certain life-history characteristics such as long lifespans and extensive pollen flow. To test this assumption, we conducted a meta-analysis with data on 97 woody plant species derived from 98 studies of habitat fragmentation. We measured the weighted response of four different measures of population-level genetic diversity to habitat fragmentation with Hedge's d and Spearman rank correlation. We tested whether the genetic response to habitat fragmentation was mediated by life-history traits (longevity, pollination mode, and seed dispersal vector) and study characteristics (genetic marker and plant material used). For both tests of effect size habitat fragmentation was associated with a substantial decrease in expected heterozygosity, number of alleles, and percentage of polymorphic loci, whereas the population inbreeding coefficient was not associated with these measures. The largest proportion of variation among effect sizes was explained by pollination mechanism and by the age of the tissue (progeny or adult) that was genotyped. Our primary finding was that wind-pollinated trees and shrubs appeared to be as likely to lose genetic variation as insect-pollinated species, indicating that severe habitat fragmentation may lead to pollen limitation and limited gene flow. In comparison with results of previous meta-analyses on mainly herbaceous species, we found trees and shrubs were as likely to have negative genetic responses to habitat fragmentation as herbaceous species. We also found that the genetic variation in offspring was generally less than that of adult trees, which is evidence of a genetic extinction debt and probably reflects the genetic diversity of the historical, less-fragmented landscape.