基因工程菌大肠杆菌JM109富集废水中镍离子的研究

利用通过基因工程技术所构建的在细胞内同时表达出高特异性镍转运蛋白和金属硫蛋白的基因工程菌富集水体中的镍离子。菌体细胞对Ni²⁺的富集速率很快,富集过程满足Langmuir等温线模型。与原始宿主菌相比,经基因改造的基因工程菌不仅最大镍富集容量增加了5倍多,而且对pH值、离子强度的变化及其它共存重金属离子的影响都呈现出更强的适应性。相比而言,Na⁺、Ca²⁺、Cd²⁺、Pb²⁺的影响较小,但Mg²⁺、Hg²⁺和Cu²⁺所引起的负面效应较大。进一步的实验表明基因工程菌对Ni2+的富集行为不需要外加营养物质。     

ApCl基因在毕赤酵母中的表达

为了研究ApCl基因的功能,应用基因重组技术将ApCl基因片段克隆入大肠埃希菌—酵母穿梭质拉pGAPZαA,构建重组真核表达质拉pGAPZαA-ApCl,电转化巴斯德单赤酵母GS115,Zeocin筛选出阳性克隆转化子.通过比较转化空载体pGAPZaA和重组载体pGAPZαA-ApCl的不同菌株分别在含有高盐和高山梨醇浓度的液体培养基中生长情况,发现毕赤酵母GS115在转化ApCl基因后其抗旱、抗盐能力显著提高(约3倍),进一步验证了ApCl基因对提高生物抗逆能力有显著作用,为将来分离该蛋白及进一步研究奠定基础.     

非人灵长类基因修饰模型研究进展

非人灵长类动物在生命科学基础研究和生物医药研究领域具有非常重要的地位。近年来随着慢病毒载体转染及靶向核酸酶(ZFN,TALEN,CRISPR/Cas9)等基因操作技术的出现,科学家们成功地获得了外源基因过表达的转基因猴和目的基因定点切割的基因编辑猴。文中对目前利用慢病毒载体获得转基因猴和利用靶向核酸酶获得基因编辑猴的研究进展进行了综述,并讨论了基因修饰猴的嵌合体现象、脱靶现象及非人灵长类动物较长性成熟时间这几个影响非人灵长类基因修饰模型推广应用的因素,最后展望了非人灵长类基因修饰模型构建技术的研究热点及发展趋势。     

Inhibition of mitochondrial complex I improves glucose metabolism independently of AMPK activation

Accumulating evidences showed metformin and berberine, well‐known glucose‐lowering agents, were able to inhibit mitochondrial electron transport chain at complex I. In this study, we aimed to explore the antihyperglycaemic effect of complex I inhibition. Rotenone, amobarbital and gene silence of NDUFA13 were used to inhibit complex I. Intraperitoneal glucose tolerance test and insulin tolerance test were performed in db/db mice. Lactate release and glucose consumption were measured to investigate glucose metabolism in HepG2 hepatocytes and C2C12 myotubes. Glucose output was measured in primary hepatocytes. Compound C and adenoviruses expressing dominant negative AMP‐activated protein kinase (AMPK) α1/2 were exploited to inactivate AMPK pathway. Cellular NAD⁺/NADH ratio was assayed to evaluate energy transforming and redox state. Rotenone ameliorated hyperglycaemia and insulin resistance in db/db mice. It induced glucose consumption and glycolysis and reduced hepatic glucose output. Rotenone also activated AMPK. Furthermore, it remained effective with AMPK inactivation. The enhanced glycolysis and repressed gluconeogenesis correlated with a reduction in cellular NAD⁺/NADH ratio, which resulted from complex I suppression. Amobarbital, another representative complex I inhibitor, stimulated glucose consumption and decreased hepatic glucose output in vitro, too. Similar changes were observed while expression of NDUFA13, a subunit of complex I, was knocked down with gene silencing. These findings reveal mitochondrial complex I emerges as a key drug target for diabetes treatment. Inhibition of complex I improves glucose homoeostasis via non‐AMPK pathway, which may relate to the suppression of the cellular NAD⁺/NADH ratio.     

2010年山东省环境污水中脊髓灰质炎病毒的监测及其基因特征分析

本研究在山东省开展了脊髓灰质炎病毒(Poliovirus,PV)的外环境监测,从济南、临沂两地采集污水标本,浓缩处理后进行病毒分离,对分离到的PV采用中和试验进行血清定型,并对其VP1及3D区进行序列测定,分析其基因突变和重组情况。2010年,共采集污水标本32份,PV阳性10份,阳性率31.3%;分离到18株PV(PV1型3株,PV2型9株,PV3型6株),均为疫苗相关株,VP1完整编码区核苷酸变异数在0~4个之间,在3株PV2型病毒和4株PV3型病毒的基因组中发现重组;对VP1区影响神经毒力的减毒位点分析发现,PV1型病毒中有1株在nt 2 749发生突变(A→G),PV2型病毒中有1株在nt2 908发生A→G突变,3株在nt2 909发生U→C突变,6株PV3型病毒全部在nt2 493发生C→U突变。环境污水中可以分离到PV,其基因重组率和主要减毒位点的回复突变率较高,未发现脊灰野毒株和疫苗衍生株脊灰病毒(Vaccine-derived poliovirus,VDPV)。     

Biodegradation of poly(ε-caprolactone) (PCL) by a new Penicillium oxalicum strain DSYD05-1

In this study, fungi isolated from soil were screened for their ability to form clear zones on agar plates with emulsified poly(ε-caprolactone) (PCL). The most active strain, designated as DSYD05, was identified as Penicillium oxalicum on the basis of morphological characteristics and phylogenetic analysis. Mutant DSYD05-1, obtained by ultraviolet-light mutagenesis from strain DSYD05, was more effective in PCL degradation. In liquid cultures of the mutant strain with PCL emulsion, DSYD05-1 showed the highest PCL-degrading activity after 4?days of cultivation. The products of PCL degradation were analysed by mass spectrometry; the results indicated that 6-hydroxyhexanoic acid was produced and assimilated during cultivation. The degradation of PCL film by DSYD05-1 was observed by scanning electron microscopy, and was indicative of a three-stage degradation process. The degradation of amorphous parts of the film preceded that of the crystalline center and then the peripheral crystalline regions. In addition, DSYD05-1 showed a wide range of substrate specificity, with capability to degrade PCL, poly(β-hydroxybutyrate), and poly(butylene succinate), but not poly(lactic acid), indicating that the strain could have potential for application in the treatment or recycling of bio-plastic wastes.     

水稻野败型细胞质雄性不育系和其保持系蛋白质多肽的比较研究

应用单向SDS—PAGE结合蛋白质铬银染色技术对水稻野败型细胞质雄性不育系珍汕97A和其保持系的叶绿体、线粒体和细胞质的蛋白质多肽进行了比较研究,发现两系之间存在明显的差异,生殖器官(穗子)上的差异比营养器官(叶片)上的差异更为显著。在成熟穗上,叶绿体可溶性蛋白不育系有25条带,保持系仅16条带,两者间有19个多肽不同;线粒体可溶性蛋白不育系有28条带,保持系比不育系少30.1和21.8KD两个多肽;细胞质可溶性蛋白丙酮沉淀物的水溶性蛋白组分不育系有24条带,保持系为29条带,两系间却有7条多肽存在差别;细胞质可溶性蛋白丙酮沉淀物的SDS-增溶性蛋白组分不育系有18条带,保持系只有11条带,两者间亦有7条多肽出现差异。由此可以看出,水稻野败型CMS表型的表达可能需要较多个基因的启动和关闭,既与叶绿体和线粒体有关,还涉及到核基因组的作用。     

重金属抗性菌HQ-1生物吸附镉与银的比较研究

从北京市远郊东三岔地区一处废弃的铅锌尾矿中筛选到一株对铅、锌、铜、钴、银、镉等重金属均有很好抗性的蜡状芽胞杆菌Bacillus cereus HQ-1。特别是对镉的抗性高达1200mg/L,具有很高的应用价值。比较研究了HQ-1菌株吸附重金属银和镉的情况与机理,发现该菌株对两种重金属离子都有较强的吸附能力。对镉的吸附行为符合Langmuir模型,对银的吸附行为符合Freundlich模型。通过红外光谱和X射线能谱对其吸附机理做了初步推断。并通过质粒消除试验证明该菌株的重金属抗性与抗性质粒存在有关。     

棕色棉F3'H-羟化酶基因的克隆与生物信息学分析

根据葡萄的类黄酮3′-羟化酶(F3'H)基因全长cDNA序列Blast所得棉花的EST序列设计引物,以开花后16 d(DPA16)的新彩棉5号(xC-5)纤维为材料,利用RACE和RT-PCR技术分离得到了2个类黄酮3′-羟化酶基因cDNA序列,此2个序列编码区完全相同,仅在3'UTR区存在片段长短的差异,推测可能是基因转录后加工方式不同所造成.克隆所获得的棉花F3'H基因编码区全长1 533 bp,编码510个氨基酸,氨基酸序列分析预测表明,该基因所编码蛋白含有一个跨膜结构域,是一种分泌蛋白,定位于内质网上,并含有一段与细胞色素P450功能区相匹配的保守功能域;序列比对结果表明,棉花F3'H基因与其他多个物种的F3'H基因在氨基酸序列上有较高的同源性;聚类分析结果表明,棉花F3'H蛋白与双子叶植物大豆的F3'H亲缘关系较为接近,而与单子叶植物高梁等作物则较远.