肝组织特异表达人核受体nr5a2转基因小鼠的构建

人乙肝病毒增强子ⅡB1结合因子（hB1F）系Ftz—F1（NR5A）亚家族的新成员。经基因重组法将人hb1 fcDNA置于小鼠白蛋白增慢子／启动子序列下游构建成肝特异重组载体,通过原核显微注射将该载体导入小鼠受精卵原核,经注射且状态良好的卯回输至假孕母鼠输卯管。产下仔鼠经PCR和Southern blotting鉴定,同时RT—PCR和Western blotting分析转基因的表达。阳性Founder鼠与正常C57鼠交配以建立转基因纯系小鼠,F1代以PCR法鉴定。结果共获得4只PCR鉴定转基因阳性Founder鼠,其中一只同时经Southern blotting鉴定为阳性。RT—PCR和Western blotting结果显示,外源基因在转基因小鼠的肝组织成功表达。遗传学分析表明,转基因已整合入小鼠基因组并可稳定溃传。     

恶臭假单胞菌NA-1菌株烟酸羟基化酶活性的诱导和转化条件的研究

恶臭假单胞菌NA-1菌株的培养和产酶特性与已报道的产酶菌株粘质沙雷氏菌(Serratiamarcescens)IFO12648和荧光假单胞菌(Psudomonasfluorescens)TN5有所不同,主要反映在最适碳源及浓度、最适诱导剂浓度和最适培养温度等方面。最适的转化条件是温度为30℃,pH为7.0,烟酸的浓度为3%。采用初步优化后的条件和流加底物的方式进行4L上罐生产,恶臭假单胞菌NA-1菌株的6-羟基烟酸产率可达到108.39gL。     

影响小鼠体细胞脂质体法转染效率的因素

We studied the effects of the amount of liposome and plasmid, exposure time of cells to the liposome-plasmid complexes, number of cell passages and cell types on GFP gene transfection of mouse somatic cells. The maximal GFP transgene expression (30.7%) was achieved when mouse fetal fibroblast cells (MFFC) at 70%-90% confluence of passage 3 were exposed for 6 h to the complexes of 4 microg liposome (LipofectAMINE) and 0.3 microg plasmid (pEGFP-N1). Under these conditions, we compared the effect of the number (from primary to 15) of passages on the transfection efficiency of MFFC. The transfection efficiency of MFFC was 10.0%, 28.9% and 7.2% at the primary, 3rd and 15th passage, respectively, which indicated that the transfection efficiency decreased with passaging. When MFFC, mouse oviductal epithelial cells (MOEC) and mouse granulosa cells (MGC) were transfected at passage 3, the transfection efficiency was 27.8%, 13.7% and 14.2%, respectively, under the described transfection conditions. When the cell cycle stages of different cell types at transfection were examined, it was found that 17.2% of MFFC, 8.7% of MOEC and 9.9% of MGC were at M phases of the cell cycle. Examination of the cell cycle stages of MFFC at different passages showed that MFFC at the third passage had the highest percentage of M cells and the percentage decreased afterwards. This suggested that the transfection efficiency was correlated with the percentages of cells at M phase, and provided essential data for improvement of the transfection efficiency.     

Improving the Activity and Stability of GL-7-ACA Acylase CA130 by Site-Directed Mutagenesis

In the present study, glutaryl-7-amino cephalosporanic acid acylase from Pseudomonas sp. strain 130 (CA130) was mutated to improve its enzymatic activity and stability. Based on the crystal structure of CA130, two series of amino acid residues, one from those directly involved in catalytic function and another from those putatively involved in surface charge, were selected as targets for site-directed mutagenesis. In the first series of experiments, several key residues in the substrate-binding pocket were substituted, and the genes were expressed in Escherichia coli for activity screening. Two of the mutants constructed, Y151αF and Q50βN, showed two- to threefold-increased catalytic efficiency (k_cat/K_m) compared to wild-type CA130. Their K_m values were decreased by ca. 50%, and the k_cat values increased to 14.4 and 16.9 s⁻¹, respectively. The ability of these mutants to hydrolyze adipoyl 6-amino penicillinic acid was also improved. In the second series of mutagenesis, several mutants with enhanced stabilities were identified. Among them, R121βA and K198βA had a 30 to 58% longer half-life than wild-type CA130, and K198βA and D286βA showed an alkaline shift of optimal pH by about 1.0 to 2.0 pH units. To construct an engineered enzyme with the properties of both increased activity and stability, the double mutant Q50βN/K198βA was expressed. This enzyme was purified and immobilized for catalytic analysis. The immobilized mutant enzyme showed a 34.2% increase in specific activity compared to the immobilized wild-type CA130.     

植物激素信号之间的相互作用

植物激素问的相互作用对植物的正常发育来说非常重要。不同植物激素之间存在相互协同、对抗和因果等关系,以精细调控植物的发育和对环境的反应等,植物激素信号之间的相互作用已成为植物细胞中不同信号间相互作用机制研究的模式系统。现对不同植物激素在生物合成、代谢、运输和信号转导途径等层次上的相互作用进行综述,并对这一领域的研究进行了总结和展望。     

鬼鹗甘肃亚种繁殖期叫声研究

利用叫声回放和声谱分析对莲花山自然保护区鬼鹗甘肃亚种（Aegolius funereus beickianus）繁殖期的叫声进行了研究。其记录了领域叫声、尖叫声等6种叫声,分析了各种叫声的特征及与行为的联系。发现不同地点录制的领域叫声存在差异,但同一个体的叫声电有变化。甘肃亚种的领域叫声和欧洲的指名亚种A.f.funereus及北美的亚种A.f.richardsoni相比较,单音数量少,单音长度小,频率高,但差异不大。     

紫外辐射增强对植物糖代谢的影响

综述了UV-B辐射增强对植物叶片、茎、根、果实以及籽粒中糖含量影响的研究现状与动态,从生理学角度分析了UV-B辐射对植物糖含量和糖代谢相关的一些重要反应及其影响植物糖含量和糖代谢的关键酶的响应,并从植物的光合碳固定、糖的合成与分解等方面阐述了UV-B影响糖含量及糖代谢的可能机理。展望了今后紫外辐射增强对植物糖代谢影响的研究重点和研究方向。     

不同植物群落下酸化尾矿养分状况及土壤酶活性

调查不同植被群落下铜尾矿的养分状况及土壤酶活性的变化动态,通过常规方法测定了尾矿的pH和电导率,以及有机质,总氮,有效磷,速效钾的含量。用底物反应法测定了过氧化氢酶,芳基硫酯酶,脲酶,酸性磷酸化酶和脱氢酶的活性。结果表明,与酸化的裸露尾矿相比,定居其上的芦苇(Phragmites australis)、狗牙根(Cynodon dactylon)和双穗雀稗(Paspalum distichum)均能显著增加尾矿基质pH值(从3.6上升到5.4),降低电导率,减缓尾矿酸化过程。芦苇和狗牙根能够显著提高尾矿基质中N、K和有机质含量(P<0.05),增加尾矿中的养分。3种植物群落下有效磷较裸露尾矿没有显著增加。植物的定居显著提高了尾矿中过氧化氢酶、芳基硫脂酶和脲酶的活性(P<0.05);但酸性磷酸化酶和脱氢酶活性没有显著提高。研究表明,植物对尾矿有明显的改良作用,而且芦苇和狗牙根优于双穗雀稗。在各种植被条件的尾矿中除酸性磷酸化酶以外的其它实验土壤酶活性均与尾矿中有机质和总氮呈极显著正相关(P<0.01)。     

两个水稻GDP-甘露糖-3’，5＇-异构酶基因特征及表达模式的研究

GDP-甘露糖-3’,5’-异构酶（GME）可以催化GDP-甘露糖转化为左旋GDP-半乳糖,该反应对于高等植物体内抗坏血酸的合成是非常重要的．但目前在分子水平上还没有对GME基因进行研究的报道．通过逆转录PCR（RT-RCR）技术从水稻成熟叶片中克隆到两个GME基因的cDNA序列,并与其他植物物种中的GMEs进行比对,结果显示,GME基因在所有植物物种中高度保守,尽管进化树分析表明单子叶植物GMEs和双子叶植物GMEs在进化上相互独立．同时,分析这两个水稻GME基因的剪切模式揭示了二者也存在高度相似性．采用半定量RT-PCR技术对两个GME基因在不同组织和不同胁迫条件下的表达模式进行研究表明,OsGME1基因在冷胁迫条件下表达水平上调,这和先前水稻冷胁迫蛋白质组学研究的结果是一致的．而OsGME2和OsGME1基因在用赤霉素处理条件下表达水平均下调,暗示赤霉素可能通过调节GME基因的表达来调控植物体内的抗坏血酸合成．