运动对2,3,7,8-TCDD持续染毒大鼠肝脏LXRa蛋白、ACC1、FAS、SCD1 mRNA表达的影响

研究运动对2,3,7,8-四氯二苯并二噁英(2,3,7,8-TCDD)持续染毒大鼠肝脏脂质合成代谢关键酶乙酰辅酶A羧化酶1(ACC1)、脂肪酸合成酶(FAS)、硬脂酰辅酶A去饱和酶1(SCD1)m RNA及转录因子肝X受体a(LXRa)蛋白表达的影响,探讨环境污染物引发代谢性疾病的发病机制,为运动锻炼防控环境健康风险提供理论支持。将24只8周龄雄性大鼠随机分为对照组(C组)、染毒组(T组)、运动染毒组(ET组)。T、ET组腹腔注射首剂量6.4μg·kg-1(以单位体重计)的2,3,7,8-TCDD,之后每隔1周给予上述剂量的21%持续染毒,连续7周。ET组尾部负重(5%体重)进行游泳运动,每周5 d,每次30 min。8周后处死动物,计算肝脏相对重量,检测肝脏甘油三酯(TG)含量,实时荧光定量PCR检测肝脏ACC1、FAS、SCD1 m RNA表达,免疫印迹法(Western Blot)检测肝脏LXRa蛋白表达。结果显示8周2,3,7,8-TCDD持续染毒可显著增加肝脏脂质合成代谢关键酶ACC1、FAS、SCD1 m RNA及转录因子LXRa蛋白表达,8周游泳运动可显著降低染毒大鼠ACC1、FAS、SCD1 m RNA及LXRa蛋白表达。上述结果表明2,3,7,8-TCDD可以引起大鼠肝脏LXRa蛋白表达增高,进而LXRa通过调控靶基因ACC1、FAS、SCD1 m RNA的表达,造成脂质代谢紊乱,肝脏甘油三酯沉积,而有氧运动降低了肝脏中脂质的沉积,提示运动干预可以改善二噁英类污染物造成的肝脏脂质代谢紊乱。     

海洋破囊壶菌△~5-延长酶和△~4-脱饱和酶在酿酒酵母中的共表达

二十二碳六烯酸(Docosahexaenoic acid,DHA C22:6n-3)是具有各种重要生理功能的高度不饱和脂肪酸.分别以质粒pYTFD5和pYFAD4为模板,扩增获得860 bp的△5-延长酶基因(elo5)和1 600 bp的△4-脱饱和酶基因(fad4).利用重叠延伸PCR构建elo5-fad4融合基因,Hind Ⅲ/Sph Ⅰ双酶切后连接到经同样处理过的pYES2.0载体,构建重组表达质粒pYEL05-FAD4.转化酿酒酵母尿嘧啶缺陷型菌株INVScl,通过缺少尿嘧啶的选择性培养基筛选阳性克隆子.添加外源脂肪酸C20:5底物.半乳糖诱导表达.气相色谱-质谱分析表明,重组酵母总脂肪酸中出现了DHA(二十二碳六烯酸,C22:6n-63)新产物,融合基因elo5-fad4在酿酒酵母中得到了表达.图7表1参18     

基于回补途径的TCA循环改造对克雷伯氏菌生长和甘油代谢的影响

回补途径和TCA循环在克雷伯氏菌(Klebsiella pneumoniae)的中心代谢中扮演着十分重要的角色.通过过表达回补途径的关键酶磷酸烯醇式丙酮酸羧化酶(PPC)和柠檬酸(TCA)循环的关键酶柠檬酸合成酶(GLTA)将磷酸烯醇式丙酮酸(PEP)和乙酰辅酶A节点的碳流引入TCA循环,以考察基于回补途径的TCA循环强化对K. pneumoniae生长和甘油代谢的影响.结果显示:单独过表达ppc或gltA基因,TCA循环还原和氧化分支的中间代谢产物琥珀酸和α-酮戊二酸分别增加了8.3倍和1.2倍,甘油利用能力明显增强,除2,3-丁二醇外,其他副产物积累量均有所下降,但1,3-PDO产量分别降低了23.3%、5.9%.共表达ppc和gltA基因后,与对照菌相比,生物量降低了35.7%,甘油利用能力进一步增强,1,3-丙二醇产量提高了10.2%,所有副产物积累量均有所减少;与单独过表达ppc或gltA基因相比,乙醇、乳酸、乙酸积累量未有明显变化,但生物量略有提高,2,3-丁二醇积累量显著降低.上述结果表明通过共表达磷酸烯醇式丙酮酸羧化酶和柠檬酸合成酶强化TCA循环能够提高菌株的甘油利用能力,弱化副产物合成,强化1,3-丙二醇的合成.(图4表2参19)     

产琥珀酸重组大肠杆菌的构建和发酵性能

琥珀酸是一种重要的化工产品,微生物发酵法生产琥珀酸具有广阔的应用前景.野生大肠杆菌厌氧发酵产物中琥珀酸含量低,且有大量副产物.为构建高产琥珀酸的重组大肠杆菌,阻断代谢旁路,利用Xer/dif重组酶系统对Escherichia coli CICIM B0013进行代谢途径的调控研究.结果显示,敲除了包括乙酸激酶和磷酸乙酰转移酶基因(ackA-pta)、乳酸脱氢酶基因(ldhA)、丙酮酸甲酸裂解酶基因(pflB)、乙醇脱氢酶基因(adhE)等琥珀酸合成竞争途径中关键酶基因的重组大肠杆菌菌株E.coli CICIM B0013-1040,其发酵液中琥珀酸成为主要产物得到积累;进一步对其PTS系统进行修饰并适当增强PEP羧化酶基因(ppc)表达剂量,成功获得一株具备较强琥珀酸生产能力的菌株E.coli CICIM B0013-1050(pTH-ppc),该菌株厌氧转化葡萄糖36 h,琥珀酸产量达到36.2 g/L,生产强度为1.01 g L-1 h-1,葡萄糖-琥珀酸转化率为64.3%,发酵液经高效液相色谱(HPLC)检测,无乳酸、乙酸、甲酸、乙醇生成.     

少根紫萍淀粉合成关键基因对寡营养胁迫的响应

浮萍是一种形态高度退化的开花植物,环境适应性广,生长条件适宜时可以接近指数增长的速度在16-48 h内实现生物量的翻倍.以寡营养处理,少根紫萍(Landoltia punctata)淀粉含量可在7 d内提高到45%以上(干重).通过深入分析转录组数据,分别挖掘到ADP葡萄糖焦磷酸化酶编码基因(LpAGP)5个、颗粒结合型淀粉合酶基因(LpGBSS)2个、可溶性淀粉合酶基因(LpSSS)2个、淀粉分支酶基因(LpSBE)5个、异淀粉酶基因7个(LpISA)、普鲁兰酶基因(LpPUL)1个.转录组定量结果表明LpAGPS1、LpAGPL2、LpAGPL3、LpGBSSI、LpGBSSII、LpSBEI-1、LpISA3和LpPUL1的表达量均因寡营养处理而上调.通过qRT-PCR检测LpAGPs等16个淀粉合成关键基因的表达量,发现绝大部分淀粉合成相关基因表达量上调,而参与淀粉降解途径相关基因(如α-淀粉酶和β-淀粉酶编码基因)的表达量因寡营养处理而下调.这种不同的调节方式促使少根紫萍淀粉合成"开源节流",淀粉得以快速积累.本研究结果可为进一步研究少根紫萍淀粉合成关键基因功能及淀粉积累机制奠定基础.     

拟南芥硫酯酶基因(atfata)在大肠杆菌中的表达及其对游离脂肪酸合成的影响

硫酯酶催化脂酰ACP(酰基载体蛋白)水解生成游离脂肪酸和ACP载体蛋白,能够解除脂肪酸合成与磷脂合成的偶联,是获得游离脂肪酸的关键基因.将拟南芥的硫酯酶基因atfata克隆到原核表达载体pET30a上,在大肠杆菌BL21(DE3)中成功表达了His-tag融合蛋白,经SDS-PAGE检测获得特异性表达条带.对该重组菌株进行摇瓶发酵实验,GC-MS检测表明其与对照菌株脂肪酸组成并未发生变化;但其脂肪酸产量达到232.06 mg/L,比对照菌株提高了70%;同时胞外游离脂肪酸产量达到了33 mg/L,占总脂肪酸含量的15%,是野生菌株胞外脂肪酸产量的7倍.图3表2参17     

一株生物素依赖型菌株bio-5:分离,初步鉴定及其用于生物素浓度测定

从西红柿根圈土壤中分离纯化到一株生物素依赖型菌株bio - 5 ,经测定其内源抗生素抗性和脂肪酸特征 ,初步定名为假单胞菌bio - 5 .这一菌株对生物素非常敏感 .建立了以此菌株用于测定生物素浓度的标准方法 .利用这一方法 ,测定了不同作物叶子中生物素的含量 ,并将这一方法成功用于筛选转stv基因烟草植物 .与传统生物素测定菌株LactobacillusplantarumATCC80 14比较 ,我们所分离的菌株具有易于获得 ,易于培养 ,快速用于生物素浓度测定的特点 .图 7表 2参 2 2     

基于RNA干扰的家蝇几丁质酶2的幼虫转录组测序

几丁质酶是昆虫几丁质降解过程中的重要酶类,在昆虫整个生长发育过程中发挥着重要作用.为筛选及挖掘与防治有害医学昆虫相关的分子靶标,根据家蝇几丁质酶2(Musca domestica chitinase 2,MdCht2)基因序列设计并合成双链RNA(double-stranded RNA,dsRNA),采用微量注射法向家蝇2龄幼虫导入dsRNA,对对照组及干扰组的样本进行转录组测序,筛选差异表达基因,进行GO(gene ontology)功能注释和KEGG(kyoto encyclopedia of genes and genomes)通路富集分析.结果显示,注射dsRNA 24 h后,幼虫MdCht2 mRNA的表达显著下降,提示导入的dsRNA干扰了MdCht2的表达.经转录组测序,与对照组相比,显著差异基因共213条,其中78条基因为上调表达,135条基因为下调表达.随机选取8条显著差异基因通过实时荧光定量PCR(qRT-PCR)进行验证,该结果与转录组结果趋势一致.根据功能注释发现这些差异基因与生长发育、脂类代谢、免疫调控等途径相关.本研究通过RNA干扰和转录组测序技术处理家蝇获得大量差异基因,结果可为后续几丁质酶相关基因的挖掘和鉴定及功能研究提供一定的数据基础.(图7表4参46)     

甘蔗光合系统Ⅰ亚基O基因的克隆与表达分析

光合系统Ⅰ亚基O(PhotosystemⅠsubunit O,Psa O)是光合系统Ⅰ中的蛋白亚基,在两个光合系统之间平衡激发能方面起着重要的作用.为研究Psa O基因的结构和功能,对甘蔗(Saccharum offi cinarum L.)叶片全长c DNA文库进行测序,获得光合系统Ⅰ亚基O基因的全长c DNA序列,命名为Sc Psa O(Gen Bank Accession Number:KF714498).生物信息学分析表明,该基因全长708 bp,开放阅读框435 bp,编码144个氨基酸;该基因所编码的蛋白定位于叶绿体基质,无信号肽,为疏水性非分泌碱性蛋白,二级结构多为无规则卷曲,含有PJN00046家族的保守结构域,参与能量新陈代谢及脂肪酸新陈代谢.同时,该基因在不同物种间具有较强的保守性,其中与同属C4植物的同源性较C3植物高.荧光定量PCR分析结果表明,甘蔗Sc Psa O基因在叶片中的相对表达量最高,具有一定的组织特异性;在氯化钠(Na Cl)、聚乙二醇(PEG)、氯化铜(Cu Cl2)、脱落酸(ABA)、水杨酸(SA)和茉莉酸甲酯(Me JA)等外源胁迫下,其表达量均呈下调趋势,且以PEG胁迫下的下调表现最为明显.这些结果表明这几种外源胁迫可能抑制甘蔗Sc Psa O基因的转录水平表达,为其进一步功能验证以及在甘蔗基因工程中的应用积累了基础资料.     

重组人核糖核酸酶抑制因子基因的siRNA表达载体构建及B16细胞中基因沉默的鉴定

人核糖核酸酶抑制因子(Human ribonuclease inhibitor, hRI)是细胞质中的一种酸性糖蛋白,可以与血管生长因子(Angiogenin, Ang)紧密结合从而抑制血管形成.利用全基因组合成法合成针对人核糖核酸酶抑制因子基因(hri)的发夹shRNA序列,亚克隆到siRNA表达载体pKD;重组载体经酶切鉴定后,用脂质体法与报告基因绿色荧光蛋白重组融合的人核糖核酸酶抑制因子的逆转录病毒载体pLNCX-EGFP-C1-hri共转染到小鼠黑色素瘤细胞B16中,在荧光显微镜下检测干扰效果.用Image-Pro plus 4.5软件对绿色荧光照片半定量分析干扰效率.结果表明,荧光显微镜显示B16中表达的绿色荧光被干扰,荧光强度半定量分析干扰效率可达80%以上.成功重组构建了针对hri的siRNA表达载体.图5参9     

桃ACC合酶基因组DNA（pACSG01）的序列分析及其表达

以桃基因组DNA为模板，用套式PCR技术扩增并克隆了桃ACC合酶基因片段，将其克隆（定名为pACSG01）并进行序列测定，表明该自然全长1320bp，并富含HindⅢ和EcoRⅠ位点，与其它ACC合酶基因结构序列有一定的相似性，内含有两个内含子，编码的氨基酸序列与已克隆桃ACC合酶cDNA推导的氨基酸序列的同源性分别为57．4％和56．8％，pACSG01与我们已克隆的两个桃ACC合酶cDNA基因表达有所不同，RNA点杂交和RT－PCR结合Southern杂交分析表明，该基因在成熟和乙烯处理的果实均不表达，伤处理、LiCl和生长素处理也不能诱导核基因的表达，在衰老花瓣中也不表达，图3参18。     

5种酚类物质对中国白羽鹌鹑和中华蜜蜂的急性毒性

酚类物质作为一类主要的污染物,已引起国内外高度重视,但目前其对陆生生物的毒性研究较少。本试验探究了4-叔丁基苯酚、间甲酚、2-氯苯酚、2-甲酚、2,4-二氯苯酚这5种酚类物质对中国本土物种中国白羽鹌鹑和中华蜜蜂的急性毒性。在中国白羽鹌鹑的急性经口试验中,2-氯苯酚、2-甲酚的7 d的半致死浓度(7 d-LC50)分别为331 mg·kg~(-1)和413 mg·kg~(-1),其他3种酚类物质的7 d-LC50均大于限度值1 000 mg·kg~(-1);在中国白羽鹌鹑的急性饲喂试验中,5种酚类物质的8 d-LC50均大于限度值2 000 mg·kg~(-1);在中华蜜蜂的急性经口试验中,2-氯苯酚、2-甲酚和2,4-二氯苯酚的48 h-LC50分别为306 mg·L~(-1)、358 mg·L~(-1)和364 mg·L~(-1);在中华蜜蜂的急性接触试验中,2,4-二氯苯酚的48 h的半致死量(48 h-LD50)为2.6μg·蜂~(-1),其他4种酚类物质的48h-LD50均大于限度值100μg·蜂~(-1)。研究结果表明不同的酚类物质由于其结构不同亦表现出不同的毒性,甲酚的邻位取代比间位取代对中国白羽鹌鹑和中华蜜蜂的毒性更高,不同物种表现出了相似的规律性。5种酚类物质对我国本土物种中国白羽鹌鹑和中华蜜蜂毒性比对其他水生生物更敏感,存在良好的剂量效应关系。     

A Conservation Gap Analysis of Brazil''s Amazonian Vegetation

Vegetation types lacking protection in the existing conservation units of the nine states in the Brazilian Legal Amazon were identified, and locations were noted where these vegetation types could be protected. Maps of vegetation, protected areas, and semi-protected areas, such as Amerindian and forestry reserves, were digitized and overlaid using a geographic information system. There are 28 natural vegetation types in the Brazilian Legal Amazon. Locations of new areas for protection were selected using a minimum criterion of protecting at least one example of each vegetation type in each state (here called "vegetation zones"). There are 111 vegetation zones in the Legal Amazon, of which only 37 (33%) have some portion of their area protected. There are few protected areas in the most heavily deforested states along the southeastern fringe of the forest. In Maranhão, where 60% of the original forest had been lost by 1990, only one of 10 vegetation types is protected. Negotiating agreements with indigenous tribes, and to a lesser extent with extractivists who harvest nontimber products from the forest, represents a major opportunity to increase significantly the area and representativeness of the conservation units. Additional conservation units need to be established quickly before rapidly increasing deforestation and land prices preclude this opportunity; otherwise, some vegetation types may virtually disappear.     

不同土地利用方式下Cd、Pb复合污染对土壤酶活性的影响

为探寻合理的土地利用方式,减轻重金属污染,以上海市崇明岛为研究对象,结合室内盆栽模拟试验,研究了不同土地利用方式下Cd、Pb复合污染对土壤酶活性的影响,以期为合理规划土地功能和提高土壤健康程度提供科学依据。试验结果表明,农田、生活用地等受人类活动影响较明显的地区,其土壤酶活性低于湿地、林地、河道旁等人为扰动较少且水分充足的地区;土壤脲酶、磷酸酶和脱氢酶活性两两之间呈显著正相关(P<0.01),且土壤脲酶对Cd、Pb较敏感;土壤有机质与土壤酶活性呈显著正相关(P<0.05);Cd、Pb复合污染在低含量(Cd:0～1mg.kg-1;Pb:0～60mg.kg-1)时对土壤酶活性表现为促进作用,而高含量(Cd:0～20mg.kg-1;Pb:0～100mg.kg-1)时则表现为抑制作用。     

Superoxide dismutase in the marine sponge <Emphasis Type="Italic">Cliona celata</Emphasis>

The aim of this work is to investigate the activity of the antioxidant enzyme superoxide dismutase in the cosmopolitan sponge Cliona celata (Grant, 1826), since this enzyme has been described as a useful biomarker for marine pollution in other marine invertebrates. The quantification of the catalytic activity for superoxide dismutase is quite complex because its substrate is an unstable free radical. Several methods have been developed for this enzymatic activity determination; most of them are based on inhibition of a redox reaction involving the superoxide radical. In this work, two methods are compared, for crude sponge extracts, as far as repeatability, reproducibility and sensibility are concerned. The adrenaline oxidation method seems to be the most adequate for these determinations. Statistical treatment of the data indicates that the reference value for the specific superoxide dismutase activity in C. celata should be in the interval [0–535.5] U/mg of total protein in wet tissues, for normal populations.     

土壤酶活性及其对土壤质量的指示研究进展

土壤中所进行的生物和生物化学过程是陆地生态系统功能的基础 ,这些过程之所以能够持续进行 ,得益于土壤中酶的作用 .酶是土壤生态系统代谢的一类重要动力 ,土壤中所进行的一切生物学和化学过程都要由酶的催化作用才能完成 .虽然单一种类的土壤酶的催化作用可能是专性的 ,但土壤中酶的来源不同 ,酶的种类繁多 .土壤中已经被鉴定出的约 6 0种酶活性表明 ,土壤酶活性是与土壤质量的很多理化指标相联系的 ,酶的催化作用对土壤中元素 (包括Ｃ、Ｎ、Ｐ、Ｓ)循环与迁移有着重要作用 .历史上 ,土壤化学和物理学属性一直被用来作为表征土壤生产力和…     

Intracellular cyanobiont Richelia intracellularis: ultrastructure and immuno-localisation of phycoerythrin,nitrogenase, Rubisco and glutamine synthetase

In marine tropical or subtropical plankton the filamentous, heterocyst-forming cyanobacterium Richelia intracellularis forms a symbiosis with the diatom Rhizosolenia clevei. An ultrastructural analysis of the apex of Rhizosolenia clevei showed that the cytoplasm in that particular part of the cell was present only where the cyanobiont was located. The cyanobiont was, however, always outside the host cytoplasm. Vegetative cells as well as the heterocysts of the cyanobiont were devoid of gas vesicles and cyanophycin granules, while carboxysomes and large glycogen granules were common. The cyanobacterial cell wall apparently remained intact in both vegetative and heterocyst cells. In green excitation light the heterocysts and vegetative cells emitted a bright yellow fluorescence, indicating that both cell types possessed high concentrations of the pigment phycoerythrin (PE) commonly associated with photosystem (PS) II. The presence of this pigment in both cell types was verified by immunogold localisation. Using the same technique, the nitrogenase (dinitrogenase reductase) enzyme was shown to be exclusively present in the heterocysts, while Rubisco was localised primarily to the carboxy-somes, which were only detected in vegetative cells. Using an antiserum against the ammonia assimilating enzyme glutamine synthetase (GS), we could demonstrate very low levels of this enzyme, indicating repression of GS in the cyanobiont.     

Der einfluß von temperaturänderungen anf enzyme der fischmuskulatur. Versuche mit Rhodeus amarus

The enzyme activities estimated in the muscles of Rhodeus amarus (Bloch) may vary according to the kind of muscle, age and sex of the test individuals, as well as with the time of day, and the season. Oxygen consumption of individuals acclimated to different temperatures was tested at 22°C. At low adaptation temperatures no adaptation was found; at high adaptation temperatures, however, compensation could be ascertained. In view of the considerable fluctuation of the enzyme activities, estimated from individuals adapted to different temperatures, a relation to certain adaptation types, established by Precht (1961b, 1968), is hardly possible. During the first days following sudden or slow changes of adaptation temperature (increase or decrease) more or less marked fluctuations of enzyme activities were obtained. Later, however, in accordance with the proceeding adaptation, a new, labile balance was established. Temperature changes represent a physiological stress even within normal temperature ranges. This fact requires general attention in studies on biological processes. Disturbance of a newly established balance of enzyme activities can be obtained, when test fishes are exposed to high temperatures. This finding may be of importance in regard to the heat resistance of the animals.     

Focal Species: A Multi-Species Umbrella for Nature Conservation

To prevent the further loss of species from landscapes used for productive enterprises such as agriculture, forestry, and grazing, it is necessary to determine the composition, quantity, and configuration of landscape elements required to meet the needs of the species present. I present a multi-species approach for defining the attributes required to meet the needs of the biota in a landscape and the management regimes that should be applied. The approach builds on the concept of umbrella species, whose requirements are believed to encapsulate the needs of other species. It identifies a suite of "focal species," each of which is used to define different spatial and compositional attributes that must be present in a landscape and their appropriate management regimes. All species considered at risk are grouped according to the processes that threaten their persistence. These threats may include habitat loss, habitat fragmentation, weed invasion, and fire. Within each group, the species most sensitive to the threat is used to define the minimum acceptable level at which that threat can occur. For example, the area requirements of the species most limited by the availability of particular habitats will define the minimum suitable area of those habitat types; the requirements of the most dispersal-limited species will define the attributes of connecting vegetation; species reliant on critical resources will define essential compositional attributes; and species whose populations are limited by processes such as fire, predation, or weed invasion will define the levels at which these processes must be managed. For each relevant landscape parameter, the species with the most demanding requirements for that parameter is used to define its minimum acceptable value. Because the most demanding species are selected, a landscape designed and managed to meet their needs will encompass the requirements of all other species.     

添加剂对绿色木霉JQF-04纤维素酶热稳定性的影响

如何提高酶蛋白的热稳定性是分子生物学、微生物学、生化工程学等学科长期所关注的重要研究课题之一.本文研究了多种添加剂对绿色木霉纤维素酶热稳定性的影响.在60℃的溶液中,多元醇(乙二醇、甘油、赤藓糖醇、木糖醇和山梨糖醇)能提高该酶的热稳定性,随着浓度的增加,赤藓糖醇、木糖醇和山梨糖醇促进酶的热稳定性呈线性增高;适当的多元醇分子长度对该酶的热稳定性有最优的保护效应;不同浓度和不同分子量的聚乙二醇对该酶的热稳定性具有明显的影响;在无机盐中,单价金属阳离子比二价金属阳离子更能显著地提高该酶的热稳定性;酶液溶剂的改变直接影响着该酶的热稳定性,该酶在D2O溶液中比在水溶液中稳定,其酶活半衰期延长了2.6倍.研究表明,热环境使酶蛋白分子的螺旋结构发生变化而失活,但某些溶质和溶剂的存在可能通过作用于蛋白质分子的三维结构而影响该酶的热稳定性.图7参15