hKv4.3基因5′非翻译区序列S160功能分析

hKv4.3基因是形成瞬时外向钾电流Ito的主要分子基础,它在心脏和神经细胞中大量表达,但在其它组织中则未见大量表达.为了研究hKv4.3表达在基因水平的调节,将hKv4.3基因的5′非翻译区的一段序列( 2~ 160,称之为S160)克隆到报告质粒中,进行瞬时表达.发现S160对hKv4.3基因的启动子和SV40的启动子都有强烈的抑制作用,没有方向特异性,但却有位置特异性.经删除突变分析,在S160片段中发现了一个抑制元件S(GAGGGGTTAA),它位于hKv4.3基因中转录起始位点下游20~30bp处.在此基础上,用RT-PCR方法对mRNA进行定量分析,初步确认这个抑制元件对蛋白表达的抑制过程是在翻译水平上.     

hKv4.3基因5＇非翻译区序列S160功能分析

hKv4.3基因是形成瞬时外向钾电流Ito的主要分子基础,它在心脏和神经细胞中大量表达,但在其它组织中则未见大量表达。为了研究hKv4.3表达在基因水平的调节,将hKv4.3基因的5＇非翻译区的一段序列（＋2～＋160,称之为S160）克隆到报告质粒中,进行瞬时表达。发现S160对hKv4.3基因的启动子和SV40的启动子都有强烈的抑制作用,没有方向特异性,但却有位置特异性。经删除突变分析,在S160片段中发现了一个抑制元件S（GAGGGGTTAA）,它位于hKv4.3基因中转录起始位点下游20～30 bp处。在此基础上,用RT-PCR方法对mRNA进行定量分析,初步确认这个抑制元件对蛋白表达的抑制过程是在翻译水平上。     

hKv4.3最小功能启动子区与上游抑制元件分析

通过克隆hKv4.3的启动子区和相关的上游调控元件,对hKv4.3基因在转录水平的调控进行了分析.对启动子区5'端一系列的删除突变分析证明,hKv4.3基因的最小功能启动子区是位于转录起始位点附近的-156～+2bp序列.经序列分析发现,这个启动子缺乏典型的TATA-box,却存在另外3个元件,即E-box(CANNTG),CArG-box[CC(A/T)₆GG]和CACC-box(GGTGC),其中CArG-box对该启动子活性起关键作用.同时在启动子区找到一个未见报道的大小为10bp的抑制子T,删除抑制子T,则启动子活性增加1倍以上.     

苜蓿根瘤菌多拷贝固氮基因启动子对根瘤发育的抑制

以荧光素酶基因作为报道基因研究苜蓿根瘤菌nif/fix基因在根瘤发育过程中的激活,发现多拷贝的nifHDK启动子和fixABCX启动子抑制根瘤的发育和固氮活性,与nifA突变型的表型相同.用β-半乳糖苷酶基因作为报道基因得到同样的结果。但是低拷贝的nifHDK启动子或fixABCX启动子对根瘤发育和固氮活性没有影响。固氮基因启动子的多拷贝抑制效应进一步说明nifA产物除了作为共生固氮基因转录的正调节因子外,对根瘤的发育也有作用。     

肿瘤抑制因子WT1对人的诱导型一氧化氮合成酶基因的转录调节作用

将人的诱导型一氧化氮合成酶(hiNOS)启动子构建在带荧光素酶基因的载体pGL3-basic上, 构建成用荧光素酶为系统的启动子, 以研究载体p8.3iNOS. 结果显示, 肾母细胞肿瘤抑制因子(WT1)能够有效地抑制hiNOS启动子的转录; 且WT1的4个选择性剪接本的抑制效果有所不同, 其中WT1(-/-)在两种肝癌细胞(HepG2和Hep3B)中对hiNOS的表达均具有最强的抑制作用, 并且抑制效果具有剂量依赖性, 用Western blot检测结果进一步证实HepG2细胞中WT1(-/-)过量表达能下调hiNOS表达. 以上结果说明WT1在肝癌细胞中对人的hiNOS具有转录调节作用.     

人工合成杆状病毒后期启动子的探讨

本文以苜蓿丫纹夜蛾核型多角体病毒(Autographa californica Nuclear PolyhedrosisVirus,AcNPV)多角体、p10、病毒粒子衣壳和核心碱性蛋白启动子的保守序列为基础,设计合成杆状病毒后期启动子——92和96碱基的互补寡聚核苷酸,并以此重组出5株在多角体启动序列、翻译起始密码子ATG上游-92处,分别或同时插入反方向的合成和多角体启动子,以及氯霉素乙酰基转移酶(CAT)基因的AcNPV毒株,探讨了带这些启动子的CAT在草地夜蛾(Spodoptera frugiperda)细胞中的表达规律及多角体基因对其表达的影响。形成多角体的重组病毒株AcNPV-vⅪⅤⅥ~+CAT和AcNPV-vSⅪⅤⅥ~+CAT中,处于合成启动子与多角体ⅪⅤ启动顺序下游的CAT基因在细胞中的表达量,要比只在多角体启动子带动下高3倍。     

天花粉蛋白基因的克隆、序列测定及在大肠杆菌和烟草中的表达

本文利用DNA多聚酶链式反应(PCR)技术,从括楼基因组DNA中扩增并克隆了天花粉蛋白基因,核苷酸序列分析结果表明,我们克隆的是天花粉蛋白的成熟肽及N端23个氨基酸的信号的编码序列,与前人从基因组或cDNA中克隆的该基因的比较发现,其同源性为99.25％,并且证实与发表的蛋白质一级结构的序列有较大差异,将该基因克隆到大肠杆菌高效表达质粒pJLA_(502)的P_RP_L启动子下游,通过温度诱导,得到了表达产物,进一步将该基因克隆到植物中间载体pE_3的花椰菜花叶病毒35S启动子下游,应用根癌农杆菌Ti质粒介导的遗传转化系统,成功地将该基因导入了烟草基因组,获得了转基因植株,Western blotting分析结果证实,天花粉蛋白基因已在大肠杆菌和转基因烟草中表达。     

基于分子信标定量检测肿瘤细胞p21 mRNA表达变化

根据p21基因序列设计合成特异性检测p21 mRNA的分子信标, 发展了一种体外快速、定量检测p21 mRNA的新方法, 将其用于肿瘤细胞p21 mRNA表达水平的检测. 结果发现: 抑制p53表达引起CNE2细胞中p21 mRNA表达水平降低, 转染ING1诱导MCF-7细胞中p21 mRNA表达上调, 5-氟尿嘧啶处理MCF-7细胞后p21 mRNA表达水平呈浓度和时间相关性变化. 这种特异性强、操作快速、简便的新方法有望广泛用于各类样品中p21 mRNA表达水平检测.     

人类端粒DNA的分子识别及其作用机制探讨

核酸中富含短的G-碱基重复的序列可以形成一种复杂的高级结构,称为G-四链体（G-quadruplex）.在基因组中,借助生物信息学发现这类富G序列广泛分布在基因的启动子区,特别是那些参与到复制中去的基因,例如癌基因.同时发现这类序列在mRNA的5′非翻译区（5′UTR）也广泛存在.这类序列在染色体末段端粒部位的存在及功能已得到充分阐明.已知端粒富含G-碱基序列,其3′末端以单链状态存在,这使得在一些小分子的选择性作用下端粒序列很容易形成G-四链体结构,进而破坏端粒结构,影响端粒酶活性.已知端粒酶在超过85%的肿瘤中过量表达,因此,端粒酶已经成为抗癌药物设计的特殊靶点,是目前本领域的研究热点之一.已发现系列配体通过有效抑制端粒酶而表现高的抗肿瘤活性.本文主要综述了近年来端粒G-四链体分子识别及其药物靶向的最新进展,并对其作用机理做了进一步的分析和探讨.     

温敏核不育系水稻株1S及其矮秆突变SV14茎的蛋白质组学比较

采用双向凝胶电泳对温敏核不育水稻株1S和其矮秆突变体SV14的茎(穗颈下第1节和第2节)蛋白进行了分离,通过银染显色,获得了分辨率和重复性较好的双向电泳图谱.选取了26个蛋白质点采用MALDI-TOF-MS进行肽质谱指纹图分析,最终有12个蛋白质点得到了可靠鉴定.其中在SV14中相对于株1S上调的仅有OSJNBa0039C07.13 蛋白,其它蛋白均表现为下调.这些差异蛋白按照功能可分为4类: (1) 能量代谢相关蛋白;(2) 次生代谢相关蛋白;(3) 调控蛋白;(4) 未知蛋白.对光合系统Ⅱ氧延伸复合物蛋白质前体2,果糖二磷酸醛缩酶,UDP-葡糖醛酸脱羧酶对应的基因进行了半定量RT-PCR分析,发现这几个基因与蛋白质的表达不一致,可能是RNA发生了翻译后修饰而减少了蛋白表达量的结果.这些差异蛋白很可能与水稻矮化有关,为水稻矮秆基因的寻找提供了另一个有效途径.     

甘氨酸在高岭土表面的吸附和热缩合反应

采用TG/DTA、FT-IR和in situ DRIFT等技术对甘氨酸在高岭土表面的吸附和热缩合反应进行了表征, 考察了甘氨酸平衡浓度和溶液pH值对吸附行为的影响, 同时探讨了原位条件下甘氨酸的缩合反应历程. 结果表明, 溶液呈弱酸性时, 甘氨酸在高岭土上的吸附量最大, 但吸附等温线不符合Langmuir模型. 在强酸性、弱酸性和碱性溶液中, 吸附态的甘氨酸分别主要以阳离子、两性离子和阴离子形式存在. 弱酸性溶液中, 甘氨酸的—NH3+基团与高岭土表面的≡S—O−(S为Si或Al)基团之间的氢键作用是吸附的主要驱动力, 而强酸性溶液中, ≡S—O−基团的质子化, 以及碱性溶液中—NH3+向—NH2的转化, 是导致吸附量下降的主要原因. In situ DRIFT结果表明, 在110−160 ℃温区, 有明显的线式二肽形成; 随着温度升高至210 ℃时, 二肽进一步脱水, 形成环化缩合产物哌嗪二酮(DKP). 没有检测到硅酯类或铝酯类中间体的特征峰, 反应可能按氢键促进下的自缩合机理进行, 高岭土的存在使缩合反应温度有明显降低.     

Regeneration and Agrobacterium-mediated transformation of the apomictic species Eulaliopsis binata

Protocols for regeneration and Agrobacterium-mediated transformation of the apomictic species Eulaliopsis binata were developed. Initially, seeds of four genotypes of E. binata were incubated on a callus induction Murashige and Skoog (MS) basal medium supplemented with three concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D). It was found that 36.2 % of explants developed highly friable callus on medium containing 3.0 mg l^?1 2,4-D. Based on frequency of callus induction, the genotype Neixiang was selected for regeneration and transformation. Callus incubated on MS basal medium supplemented with 0.2 mg l^?1 α-naphthalene acetic acid and 6.0 mg l^?1 6-furfuryl-aminopurine developed shoots. Subsequently, Agrobacterium tumefaciens strain EHA105—harboring a plasmid pCAMBIA1381 carrying a hygromycin phosphotransferase (hpt) resistance gene and a synthetic green fluorescent protein (GFP) gene, both driven by the cauliflower mosaic virus 35S promoter—was used for transformation system. Putative transgenic callus was obtained following two cycles of hygromycin selection. Expression of the transgene(s) in putative transgenic callus was analyzed using the GFP detection. Molecular identification of putative transformed shoots was performed by polymerase chain reaction and Southern blot analysis to confirm presence and integration of the hpt gene.     

Conductometric sensor with calixarene-based chemosensitive element for the arginine detection

A possibility of the creation of conductometric chemosensor with calixarene-based sensitive element for arginine detection was evaluated. The surface of gold interdigitated electrodes of conductometric transducer was modified with calixarene. The optimal concentration of calixarene for preparation of chemosensitive element was determined to be 100 mg/ml. The basic analytical characteristics of the developed chemosensor were determined (sensitivity to arginine—37.5 μS/mM, limit of arginine detection 5 μM, linear range 0.005–150 μM, response time 150 s) and analyzed as regards its application for arginine determination. It was established for all types of developed sensors that they have good reproducibility of signals to arginine over one working day; the measurement error (RSD) did not exceed 5%. The selectivity of calixarene-based chemosensor to arginine was investigated as well as the selectivity of calixarene complexation with arginine compared with other amino acids.     

CO<Subscript>2</Subscript> Removal from Biogas by Cyanobacterium <Emphasis Type="Italic">Leptolyngbya</Emphasis> sp. CChF1 Isolated from the Lake Chapala,Mexico: Optimization of the Temperature and Light Intensity

(S)-N-Boc-3-hydroxypiperidine ((S)-NBHP) is a key pharmaceutical intermediate and the chiral source in synthesizing Imbruvica, which is a newly approved drug in lymphoma therapy by targeting Bruton’s tyrosine kinase (BTK). Current chemical synthesis of (S)-NBHP suffered from the need of noble metal catalyst and low yield. The single reported bioconversion of (S)-NBHP was achieved by using recombinant ketoreductase, but enzyme sequence was kept confidential and the catalytic process suffered from the thermodeactivation and substrate inhibition. In the current study, we presented a thermostable aldo-keto reductase (AKR)—AKR-43—which showed high activity toward N-Boc-3-piperidone (NBP) to produce (S)-NBHP, high enantioselectivity, and no substrate inhibition. The molecular simulations demonstrated the structural rationale for the enantioselectivity of AKR-43 toward NBP and supported the classic ordered two-step catalytic mechanism. The catalytic process was achieved by using glucose dehydrogenase (GDH) for cofactor recycling, and the optimal reaction conditions were determined to be 30 °C and pH 7.5. Within a reaction time of 16 h, the 16 % substrate concentration (w/w), over 99 % ee and under 3.5 % of enzyme loading (w/w) characterized a high efficiency process with promising industrial values.     

A velocity map imaging study of the one and two photon dissociations of state-selected DCl+ cations

DCl(+)(X (2)Pi(32),v(+")=0) cations have been prepared by 2+1 resonance enhanced multiphoton ionization, and their subsequent fragmentation following excitation at numerous wavelengths in the range of 240-350 nm studied by velocity map imaging of the resulting Cl(+) products. This range of excitation wavelengths allows selective population of A (2)Sigma(+) state levels with all vibrational (v(+')) quantum numbers in the range 0< or =v(+')< or =15. Image analysis yields wavelength dependent branching ratios and recoil anisotropies of the various D+Cl(+) ((3)P(J), (1)D, and (1)S) product channels. Levels with 10< or =v(+')< or =15 have sufficient energy to predissociate, forming D+Cl(+)((3)P(J)) products with perpendicular recoil anisotropies-consistent with the A (2)Sigma(+)<--X (2)Pi parent excitation and subsequent fragmentation on a time scale that is fast compared with the parent rotational period. Branching into the various spin-orbit states of the Cl(+)((3)P(J)) product is found to depend sensitively upon v(+') and, in the case of the v(+')=13 level, to vary with the precise choice of excitation wavelength within the A (2)Sigma(+)<--X (2)Pi(13,0) band. Such variations have been rationalized qualitatively in terms of the differing contributions made to the overall predissociation rate of DCl(+)(A,v(+')) molecules by coupling to repulsive states of (4)Pi, (4)Sigma(-), and (2)Sigma(-) symmetries, all of which are calculated to cross the outer limb of the A (2)Sigma(+) state potential at energies close to that of the v(+')=10 level. Cl(+)((3)P(J)) fragments are detected weakly following excitation to A (2)Sigma(+) state levels with v(+')=0 or 1, Cl(+)((1)D) fragments dominate the ion yield when exciting via 2< or =v(+')< or =6 and via v(+')=9, while Cl(+)((1)S) fragments dominate the Cl(+) images obtained when exciting via levels with v(+')=7 and 8. Analysis of wavelength resolved action spectra for forming these Cl(+) ions and of the resulting Cl(+) ion images shows that (i) these ions all arise via two photon absorption processes, resonance enhanced at the one photon energy by the various A(v(+')<10) levels, (ii) the first A (2)Sigma(+)<--X (2)Pi absorption step is saturated under the conditions required to observe significant two photon dissociation, and (iii) the final absorption step from the resonance enhancing A(v(+')) level involves a parallel transition.     

Potential of cross-priming amplification and DNA-based lateral-flow strip biosensor for rapid on-site GMO screening

The requirement to monitor the presence of genetically modified organisms (GMO) in a variety of marked products has generated an increasing demand for reliable, rapid, and time and cost-effective analytical methods. Here we report an on-site method for rapid detection of cauliflower mosaic virus promoter (CaMV 35S), a common element present in most GMO, using cross-priming amplification (CPA) technology. Detection was achieved using a DNA-based contamination-proof strip biosensor. The limit of detection was 30 copies for the pBI121 plasmid containing the CaMV 35S gene. The certified reference sample of GM maize line MON810 was detectable even at the low relative mass concentration of 0.05 %. The developed CPA method had high specificity for the CaMV 35S gene, as compared with other GM lines not containing this gene and non-GM products. The method was further validated using nine real-world samples, and the results were confirmed by real-time PCR analysis. Because of its simplicity, rapidity, and high sensitivity, this method of detecting the CaMV 35S gene has great commercial prospects for rapid GMO screening of high-consumption food and agriculture products.     

A New Aminopeptidase from the Keratin-Degrading Strain Streptomyces fradiae var. k11

An aminopeptidase gene fragment was isolated from a keratin-degrading strain, Streptomyces fradiae var. k11, by PCR amplification using a degenerate primer set designed based on the partial amino acid sequence of the native enzyme. The gene, designated sfap, encoded a polypeptide of 461 amino acids comprised of three domains: a signal peptide, a mature region, and a C-terminal propeptide. The aminopeptidase, SFAP, had highest amino acid sequence identity (79%) with a putative aminopeptidase from Streptomyces griseus subsp. griseus NBRC 13350. The gene with and without C-terminal propeptide was successfully overexpressed in Escherichia coli BL21 (DE3), and the gene without C-terminal propeptide encoded a functional enzyme. Purified recombinant SFAP exhibited optimal activity at pH 8.0 and 60 °C, and retained >60% peak activity over a broad range of temperature. The enzyme was thermal and pH stable, and showed metalloprotease characteristics, which was inhibited by EDTA but activated by Ca²⁺ and Co²⁺. This is the first study to report the gene cloning and expression of a leucine aminopeptidase from S. fradiae.     

Effects of Phytohormones and Jasmonic Acid on Glucosinolate Content in Hairy Root Cultures of Sinapis alba and Brassica rapa

Although some study have established hairy root cultures from brassicaceous plants with glucosinolates (GS) as characteristic secondary metabolite, studies are missing which compare hairy roots with the corresponding mother plants. Therefore, two different plant species—Sinapis alba and Brassica rapa subsp. rapa pygmeae teltoviensis—were transformed with the Agrobacterium rhizogenes strain A4. Aliphatic and indolyl GS were present in B. rapa, exhibiting larger quantities in leaves than in roots. Aromatic p-hydroxybenzyl GS were found particularly in the leaves of S. alba. However, the proportion of indolyl GS increased suddenly in transformed hairy roots of S. alba and B. rapa. Cultivation with the phytohormone kinetin (0.5 mg?L^?1) enhanced GS accumulation in B. rapa hairy roots, however not in S. alba, but 2,4-D (0.4 mg?L^?1) induced de-differentiation of roots in both species and reduced GS levels. GS levels especially of 1-methoxyindol-3ylmethyl GS increased in hairy roots in response to JA, but root growth was inhibited. While 2 weeks of cultivation in 100 to 200 μM JA were determined at optimum for maximum GS yield in S. alba hairy root cultures, 4 weeks of cultivation in 50 to 100 μM JA was the optimum for B. rapa.