编码水稻胞外核苷酸双磷酸酶基因的表达特性

NTPDase(Nucleoside triphosphate-diphosphohydrolase,核苷酸双磷酸酶)在哺乳动物巾分布于细胞膜,可以水解细胞外ATP和其他核苷酸,从而调控胞外核苷酸代谢及信号应答.根据水稻NTPD基因序列设计引物,在不同胁迫条件样品中,通过PCR扩增水稻NTPD基因,结果表明水稻NTPD基因表达水平在盐胁迫样品中升高.为进一步分析水稻NTPD基因的功能,构建了该基因的过量表达、RNA干涉载体,通过根癌农杆菌介导转入水稻品种日本晴并获得阳性植株.半定量RT-PCR分析达到过量表达和干涉日的.在盐胁迫条件下,TO代过表达转基因植株表现出一定程度的抗胁迫能力,RNA干涉转基因植株的生长则受到抑制.图7表3参20     

水稻锌指蛋白基因OsWIP6的克隆、表达及RNAi载体转化

为了解水稻C2H2型锌指蛋白转录因子基因的表达调控机理及生物学功能,克隆了水稻的一个C2H2型锌指蛋白转录因子基因(OsWIP6),并构建其RNA干涉植物表达载体,通过农杆菌介导转化获得转基因植株.生物信息学分析显示,OsWIP6氨基酸序列与拟南芥两个WIP家族转录因子高度同源.组织差异性表达分析表明,该基因在水稻花发育上具有表达偏向性.与野生型相比,转基因水稻表现为抽穗延迟,结实率及千粒重降低,半定量分析显示转基因植株OsWIP6表达量明显下降.因此,推断OsWIP6基因与水稻育性密切相关.     

水稻DDB1基因的表达特性及功能分析

利用RNA干涉技术构建水稻DDB1(Damaged DNA binding protein 1)基因不同启动子驱动植物表达载体,DDB1-RNAi和DDB1-glu-RNAi.通过根癌农杆菌介导转入水稻愈伤,经组织培养成功获得转基因植株.半定量RT-PCR分析显示,与野生型植株相比,两组转基因植株幼叶内DDB1的表达量...     

GCC盒和JERE盒介导的信号途径在水稻应答逆境中的作用

植物应答逆境的防御反应在很大程度是在基因转录水平调节的,其中转录因子与目标基因启动子上顺式作用元件的识别和结合起着关键的调节作用.本研究将从双子叶植物中发现的GCC盒(应答乙烯)和JERE盒(应答JA和激发子)与CaMV35S核心启动子融合构建成诱导型启动子,利用GUS报告基因构建其表达载体并进行农杆菌介导的水稻遗传转化.利用T1代株系分析了GCC盒和JERE盒水稻植株对不同逆境胁迫和激素处理的应答.结果表明在转基因植株中它们都具有很低的本底表达.稻瘟病侵染、稻纵卷叶螟取食和机械损伤处理可不同程度提高GUS基因的表达.另外,脱落酸(ABA)、水杨酸(SA)和茉莉酸(JA)也能提高GUS的表达.实验结果暗示JERE和GCC盒介导的信号途径在单子叶和双子叶之间有一定的保守性.图5参15     

番茄ARF4基因果实特异RNAi载体的构建及遗传转化

构建ARF4基因果实特异表达的RNA干涉载体,对转基因番茄果实进行初步分析,可为采用基因工程方法改良番茄果实品质做出新尝试.利用RT-PCR技术从番茄果实cDNA中扩增SlARF4基因全长,将番茄ARF4基因正反向重复序列片段导入到植物表达载体pBI121上,启动子是番茄果实特异表达的TFM7.将构建的ARF4基因果实特异RNA干涉载体pBI121-TFM7-A4Ri通过根癌农杆菌介导转入到野生型番茄中,进而对转化获得的植株进行了阳性鉴定.分别以转基因番茄和野生型番茄为材料,分析ARF4在果实中的表达水平,测定绿熟期果实叶绿素含量、果实的单果重量和果皮厚度.酶切证实pBI121-TFM7-A4Ri果实特异表达载体构建成功,而且,PCR检测也得到阳性转基因株.半定量RT-PCR分析显示,转基因番茄果实中ARF4的表达量明显低于野生型果实.转基因番茄果实的叶绿素含量、单果重量和果皮厚度都比野生型有提高.因此,ARF4果实特异表达的RNAi方法能够改良番茄果实品质.     

不同虫压下转Bt基因水稻与非转基因水稻生态适合度差异

为了解转基因水稻的基因扩散效率和潜在生态风险,以转Bt抗虫水稻Bt63、R1、R2和非转基因常规水稻Ⅱ优838为试材,采用高、低两个不同虫害胁迫水平和转基因与非转基因水稻相间种植方式,通过观察水稻植株营养生长、结实以及对螟虫危害的抗性表现等差异,研究比较了Bt外源基因插入后对水稻植株适合度的影响.结果表明:在低虫害胁迫条件下,转Bt基因水稻在植株分蘖数、生物量鲜重等营养生长指标方面与非转基因对照品系间无明显差异,但株高、穗长、穗重等指标不及对照,且R2和Bt63与对照间差异显著;在高虫害胁迫条件下,3个转Bt基因水稻品系的分蘖数、穗长、穗重等指标明显高于对照.而不同转基因品系株高适合度效应不同,这可能与受体本身的特性相关.3种转基因水稻的单株结实粒数、千粒重与对照在两种虫害胁迫条件下均无显著性差异,Bt基因对受体植株的结实影响不明显.在高虫害胁迫条件下,3种转Bt基因水稻的抗虫能力均显著优于非转基因水稻,表明Bt基因对受体植株的抗虫性影响显著.同时,本研究结果还表明转Bt基因水稻的适合度代价较小,预示了抗虫转基因水稻外源Bt基因在一定环境条件下具有逃逸的可能,但这种风险比较小.     

番茄HP1和HP2基因RNA共干涉载体的构建及遗传转化

番茄HP1和HP2是色素积累的负调控因子,在光形态建成和色素积累调控中起着重要作用.将番茄HP1、HP2基因片段导入到植物表达载体pBI121,用番茄果实特异表达的TMF7基因的启动子替换原有的CaMV 35S启动子,构建果实特异表达HP1、HP2双基因RNA共干涉植物表达载体pBI121-TMF7-HP1HP2.通过根癌农杆菌介导转入番茄子叶,经组织培养成功获得转基因植株.半定量RT-PCR分析显示,转基因植株果实内HP1、HP2的表达量均明显低于野生型植株果实.转基因植株果实叶绿素含量比野生型明显升高,而叶片中的叶绿素含量无明显差异.该研究结果为采用基因工程的方法改善番茄果实营养品质作出了新的尝试和提出了新的思路.图6表2参15     

番茄SIZ1-like1基因的克隆与功能

SUMO(Small ubiquitin-related modifi er)化修饰是植物体内一种重要的蛋白质功能调节方式.它调控植物细胞中蛋白的降解与定位,植物抗性及激素调节等.SIZ1是SUMO的E3连接酶并在SUMO化中起重要作用.为了解SIZ1基因在番茄中的功能,成功克隆番茄(Solanum lycopersicum)SIZ1基因(SIZ1-like1)并构建番茄SIZ1-like1RNA干涉和过表达载体后,通过农杆菌介导转入野生型番茄,成功获得6个独立的转基因阳性植株.荧光定量PCR(Real-time PCR)分析野生型番茄中SIZ1-like1基因的组织表达特异性,发现SIZ1-like1在植物的各个组织中都有表达.构建SIZ1-like1黄色荧光蛋白融合表达载体,通过对SIZ1-like1融合黄色荧光蛋白转基因番茄的根尖进行荧光显微观察,证实番茄SIZ1蛋白定位于细胞核.干旱胁迫实验分析显示,过表达转基因植株抗旱性强于野生型,且脯氨酸含量是野生型的3倍左右,而RNAi植株抗旱能力则较弱.因此SIZ1基因对番茄抗旱起到了正调控作用.     

水稻抗虫转基因光温敏核不育系植株的获得

以质粒pCUBAC-HPT作抗虫基因供体，优良光温敏核不育系612S为受体，采用基因枪转化法，获得了转抗虫基因sck和sbk的植株．分子证据表明，外源基因己整合到受体基因组中．蛋白活性测定和Western blotting结果显示，转基因在受体植株中得到表达．田间抗虫性测定表明，外源基因的转入提高了受体的抗虫性．本文还讨论了转基因技术在水稻育种中的作用．图5表2参12     

双农杆菌共转化获得无标记转pepc基因的水稻植株

利用双农杆菌/双质粒的共转化系统获得了无选择标记转pepc基因的水稻植株.采用两个农杆菌EHA105转化粳稻品种台粳9号幼胚诱导的胚性愈伤组织,其中一个农杆菌内的质粒表达载体的T-DNA区仅含有目的基因pepc,另一个的T-DNA区含有选择标记基因hpt和GUS报告基因.获得抗性愈伤的转化率为9.2%.85.3%的抗性愈伤分化成T0代株系,其中78.8%的T0代植株为GUS阳性转基因植株.PCR分析表明,在78个T0代GUS阳性植株(来源于23个抗性愈伤组织)中共有35个植株(来源于7个抗性愈伤组织)含有pepc基因,即在23个GUS阳性株系中发生共转化株系的频率为30.4%.7个共转化的株系中有5个株系自交产生后代植株中含有无标记转pepc基因植株,其比例为71.4%.无标记的转pepc基因水稻植株中PEPC活性平均比对照提高8.8倍;与非转基因对照植株相比,转pepc基因水稻植株表现出更强的光合能力.图5表3参26     

Removal of arsenic by Acidothiobacillus ferrooxidans bacteria in bench scale fixed-bed bioreactor system

In the present study arsenic contaminated simulated water and groundwater was treated by the combination of biological oxidation of tri-valent arsenite [As (III)] to penta-valent arsenate [As (V)] in presence of Acidothiobacillus ferrooxidans bacteria and its removal by adsorptive filtration in a bioreactor system. This method includes the immobilisation of A.ferrooxidans on Granulated Activated Carbon (GAC) capable of oxidising ferrous [Fe (II)] to ferric [Fe (III)]. The Fe (III) significantly converts the As (III) to As (V) and ultimately removed greater than 95% by the bed of GAC, limestone, and sand. The significant influence of Fe (II) concentration (0.1–1.5?gL^?1), flowrate (0.06–0.18?Lh^?1), and initial As (III) concentration (100–1000?µgL^?1) on the arsenic removal efficiency was investigated. The simulated water sample containing the different concentration of As (III) and other ions was used in the study. The removal of other co-existing ions present in contaminated water was also investigated in column study. The concentration of arsenic was found to be <10?µgL^?1 which is below Maximum Contaminant Level (MCL) as per WHO in treated water. The results confirmed that the present system including adsorptive-filtration was successfully used for the treatment of contaminated water containing As (III) ions.     

Sub-chronic exposure to carbendazim induces biochemical and hematological alterations in male goats

The present investigation reports the effect of repeated (90 days) administration of carbendazim on the biochemical and hematological parameters in male goats. Carbendazim administered orally at a daily dose of 50 mg kg^?1 body weight for 90 consecutive days resulted in increased plasma concentration of aspartate aminotransferase (AST), alanine aminotransferase (ALT), lactate dehydrogenase (LDH), gamma-glutamyl transferase (GGT), creatinine and albumin, while alkaline phosphatase (ALP) and glucose levels were decreased. Decrease in total leukocyte (TLC) and neutrophil count and increase in lymphocyte count was observed in the treatment group. The findings of the present investigation indicate that sub-chronic exposure to carbendazim in male goats causes hepatic and slight kidney dysfunctions.     

Verification of a 3D CFD model for vertical slot fish-passes

In the present work, we verified a 3D computational fluid dynamics model for vertical slot fish-passes (VSFs) that employs the renormalization-group k-epsilon turbulence model (RNG KE) and the volume of fluid (VOF) method. We compared model calculations with experiments in two pool designs T1 and T2 of an experimental VSF and with 2D calculations using the shallow water equations (SWE) and the standard k-epsilon (2D SKE) model. Calculations of the 3D model showed (1) good agreement with experiments and 2D calculations in predicting mean flow velocities, (2) better performance in the determination of the water surface in the VSF, which is attributed to the accurate VOF method, (3) superior prediction of turbulence characteristics than the 2D model, which is due to the 3D RNG KE model that overcomes the problem of turbulence overestimation of the 2D SKE model, and to the fact that the 3D model takes into account the 3D features of the flow in the fish pass. Moreover, the present 3D calculations showed that the common assumptions in VSFs that (1) the flow is 2D, and (2) the simulation of 4 pools of a VSF is sufficient to obtain satisfactory results, are not always valid. Flow can be considered as 2D only in pool design T2 and for certain geometries and flow characteristics in pool design T1; while, eventually, all the pools of a fish pass need to be modeled to ensure accurate results. Finally, the present work illustrates the need to perform fish experiments simultaneously with flow experiments.     

Impact of malathion on the biochemical parameters of gobiid fish, Glossogobius giuris (Ham)

There is a dearth of information regarding the changes in heart muscle metabolites induced by pesticides. In the present study, the gobiid fish, Glossogobius giuris, was exposed to sub lethal concentrations of (0.05, 0.25 and 0.5 ppm) organophosphorus pesticide, malathion for short duration (24 to 96 hr). The cardiac muscles showed maximum depletion of glycogen and cholesterol content during 72 and 96 hr after treatment with 0.5 ppm malathion. Whereas a slight fluctuation of protein and glycogen content was observed in low concentration (0.05 ppm) of malathion. The levels of protein showed a significant decrease at high concentration (0.5 ppm) when treated for longer duration (96 hr). The present study reports metabolic dysfunction in response to malathion toxicity in the fish.     

Ostracods as indicators of pollution in the lakes of Mysore

Ostracods are small crustaceans found in aquatic habitats and the present paper deals with the role of water quality index on their population diversity and seasonal fluctuations in the four lakes of Mysore city The present investigation was carried out from July 2004 to June 2005. The study revealed highest water quality index and population density of ostracods during summer and least during winter Dalvoi lake recorded higher waterquality index (125.04), population density (60 l(-1)) but lower species diversity (2 species) of ostracods, whereas Kamana lake recorded lowest water quality index (63.49), population density (40 l(-1)) and highest species diversity (6 species) of ostracods. Increase in the water quality index indicates increase in the pollution load. As water quality index (WQI) increases, population density of ostracods increases but species diversity decreases.     

Arsenic uptake and speciation in vegetables grown under greenhouse conditions

The accumulation of arsenic (As) by vegetables is a potential human exposure pathway. The speciation of As in vegetables is an important consideration due to the varying toxicity of different As species. In this study, common Australian garden vegetables were hydroponically grown with As-contaminated irrigation water to determine the uptake and species of As present in vegetable tissue. The highest concentrations of total As were observed in the roots of all vegetables and declined in the aerial portions of the plants. Total As accumulation in the edible portions of the vegetables decreased in the order radish ≫ mung bean > lettuce = chard. Arsenic was present in the roots of radish, chard, and lettuce as arsenate (As^V) and comprised between 77 and 92% of the total As present, whereas in mung beans, arsenite (As^III) comprised 90% of the total As present. In aerial portions of the vegetables, As was distributed equally between both As^V and As^III in radish and chard but was present mainly as As^V in lettuce. The presence of elevated As in vegetable roots suggests that As species may be complexed by phytochelatins, which limits As translocation to aerial portions of the plant.     

Chemopreventive effects of ellagic acid against genotoxicity induced by benzo(a)pyrene

Benzo(a)pyrene (B(a)P) is a commonly used indicator for polycyclic aromatic hydrocarbons (PAHs) contamination. The use of certain phytochemicals and some polyphenolic compounds proved to be effective in blocking PAH-induced effects. The aim of the present study was to examine the possible inhibitory effects of ellagic acid (EA), present in berries and nuts, against B(a)P-mediated toxicity using in vitro and in vivo test systems. In vitro method included the Ames test, using Salmonella typhimurium tester strain TA100. In vivo experiments were based on mouse bone marrow micronucleus (MN) assay and immunoblotting. EA produced a decrease in the number of revertant colonies against B(a)P in the Ames assay. Further, there was a reduction in the mean number of micronucleated polychromatic erythrocytes in the presence of EA in the co-treatment protocol of the MN test. Western blotting results showed downregulation of CYP450 isoform CYP1A1 which prevented bioactivation of B(a)P. Thus, the present study demonstrates antimutagenic role played by EA against the mutagenic activity of B(a)P in both in vitro and in vivo systems.     

Effect of photoperiod and temperature on testicular regression in Channa punctatus

In Channa punctatus testicular regression commonly observed during spawning and postspawing phases of reproductive cycle. In the present study testicular regression was frequently noticed in fish maintained under both long photoperiod-warm temperature (LD 16 : 8-30 degrees C) and short photoperiod-warm temperature (LD 8 : 16-30 degrees C) regimes. Testicular regression was characterized by distortion of cellular boundary of lobules and formation of collagenous capsules containing degenerating germ cells, blood cells and colloidal mass within the lobules. The magnitude of testicular regression was more in fish exposed to short photoperiod regime (R-73.33%, SP-41.67%) than long photoperiod regime (R-50.83%, SP-19.16%) and control group (R-20.83%, SP-16.67%) in both resting (R) and spawning (SP) phases. Further the frequency of testicular regression during resting phase was 73.33% (short photoperiod), 50.83% (long photoperiod) and 20.83% (control) whereas during spawning phase was 41.67% (short photoperiod) 19.16% (long photoperiod) and 16.67% (control). In the present study occurrence of more testicular regression during resting phase than spawning phase may be due to change in the endogenous condition of the fish.     

气态甲醛致雌性小鼠生殖细胞DNA-蛋白质交联的研究

为了研究气态甲醛对雌性小鼠生殖细胞的影响,以昆明雌性小鼠卵巢为实验材料,采取动态吸入式染毒方式,应用KCl-SDS沉淀法检测了气态甲醛染毒后所引起的小鼠卵巢生殖细胞DNA-蛋白质交联(DPC)效应.结果表明,较低浓度的甲醛(0.5mg·m-3),即可导致明显的DPC效应(与对照相比,p<0.05),并且随着染毒浓度的升高(0.5、1.0、3.0mg·m-3),DPC系数也逐渐升高.上述结果表明在实验所设浓度范围内,甲醛对小鼠卵巢生殖细胞的DNA损伤可以引起DNA-蛋白质的交联,并且DPC系数随着染毒浓度的增大而增大.DNA-蛋白质交联是DNA分子的一种严重损伤,气态甲醛对雌性小鼠生殖细胞具有一定的影响.     

Tributylzinn auf dem Weg zu den ‘Top of the POPs’?

Two groups of pollutants established during the past years, the impact of which on the eco-system gave cause for concern and legislative reaction: Persistent Organic Pollutants (POPs) and Endocrine Disrupters. At present, only the 12 POPs (‘Dirty Dozen’) are subject to serious international bans as long as they do not belong as well to potential endocrine disrupters, such as the PCDDs/PCDFs (Polychlorinated Dibenzo(p)dioxins and furans). But there are endocrine disrupters which fulfil, the POP-criteria stated in the Stockholm Convention. The organotin compounds are part of them, especially the tributyltin (TBT), since the toxicity increases with increasing alkylation. The present investigations may contribute to the discussion on the POP-potential of TBT on the basis of the results from GfA-internal and literature findings.