大鼠肝大部分切除前热休克对热休克蛋白和磷酸酶的影响*

定性和定量分析了在大鼠2/3肝切除前8h热休克(46℃,30min)处理(HS-PH)的肝再生期间(0-144h)保持性热休克蛋白70/诱导性热休克蛋白68(HSC70/HSP68)分布和含量变化、酸性磷酸酶(ACP)和碱性磷酸酶(AKP)分布、种类和活性变化.并把上述结果同只进行热休克(46℃,30min)(HS)和只进行2/3肝切除(PH)时这些分子的变化进行了比较.发现三种处理均可提高ACP、AKP活性和HSC70/HSP68表达量,但它们的变化规律不同.进一步分析发现,HS-PH后ACP活性增强是与140kD酶活性增加有关,而AKP活性增强则与140和160-180kD的酶活性增加有关.根据实验结果推测,ACP、AKP和HSC70/HSP68均在肝细胞的热休克反应和肝再生中起作用;它们可能均参与这些过程中的信号传导,但ACP可能在启动肝细胞增殖中起主导作用,AKP和HSC70/HSP68可能在胞质分裂中起主导作用.     

小麦茎顶端原基分化的综合模式

研究了小麦(Triticum aestivum L.)茎顶端不同类型原基分化的动态过程,以明确原基分化的综合模式,并建立了不同原基分化之间的定量关系.结果表明,小麦叶原基和苞叶原基分化与播后累积生长度日(GDD, growing degree days after sowing)的关系呈S形曲线,而小穗原基和小花原基为上升段抛物曲线.从分化模式看,苞叶原基具备营养器官原基特征;小穗和小花原基的分化进程能较好地反映基因型和生态条件对顶端发育的影响.小麦茎顶端原基分化的综合模式为由三段子模式构成的近似S曲线.叶原基数由基因型和环境条件共同决定,而苞叶原基、小穗原基和小花原基数以环境因子的影响为主.以平均热间距来衡量,适期播种处理的叶片、苞叶和小穗原基分化速率最高;而小花原基数与小花分化持续期之间的数量关系最为密切.研究结果有助于揭示和理解小麦茎顶端发育的生物学规律.     

Arabidopsis CALCIUM-DEPENDENT PROTEIN KINASE8 and CATALASE3 Function in Abscisic Acid-Mediated Signaling and H2O2 Homeostasis in Stomatal Guard Cells under Drought Stress

Drought is a major threat to plant growth and crop productivity. Calcium-dependent protein kinases (CDPKs, CPKs) are believed to play important roles in plant responses to drought stress. Here, we report that Arabidopsis thaliana CPK8 functions in abscisic acid (ABA)- and Ca²⁺-mediated plant responses to drought stress. The cpk8 mutant was more sensitive to drought stress than wild-type plants, while the transgenic plants overexpressing CPK8 showed enhanced tolerance to drought stress compared with wild-type plants. ABA-, H₂O₂-, and Ca²⁺-induced stomatal closing were impaired in cpk8 mutants. Arabidopsis CATALASE3 (CAT3) was identified as a CPK8-interacting protein, confirmed by yeast two-hybrid, coimmunoprecipitation, and bimolecular fluorescence complementation assays. CPK8 can phosphorylate CAT3 at Ser-261 and regulate its activity. Both cpk8 and cat3 plants showed lower catalase activity and higher accumulation of H₂O₂ compared with wild-type plants. The cat3 mutant displayed a similar drought stress-sensitive phenotype as cpk8 mutant. Moreover, ABA and Ca²⁺ inhibition of inward K⁺ currents were diminished in guard cells of cpk8 and cat3 mutants. Together, these results demonstrated that CPK8 functions in ABA-mediated stomatal regulation in responses to drought stress through regulation of CAT3 activity.     

Mutation Spectrum of Six Genes in Chinese Phenylketonuria Patients Obtained through Next-Generation Sequencing

Background

The identification of gene variants plays an important role in the diagnosis of genetic diseases.

Methodology/Principal Findings

To develop a rapid method for the diagnosis of phenylketonuria (PKU) and tetrahydrobiopterin (BH4) deficiency, we designed a multiplex, PCR-based primer panel to amplify all the exons and flanking regions (50 bp average) of six PKU-associated genes (PAH, PTS, GCH1, QDPR, PCBD1 and GFRP). The Ion Torrent Personal Genome Machine (PGM) System was used to detect mutations in all the exons of these six genes. We tested 93 DNA samples from blood specimens from 35 patients and their parents (32 families) and 26 healthy adults. Using strict bioinformatic criteria, this sequencing data provided, on average, 99.14% coverage of the 39 exons at more than 70-fold mean depth of coverage. We found 23 previously documented variants in the PAH gene and six novel mutations in the PAH and PTS genes. A detailed analysis of the mutation spectrum of these patients is described in this study.

Conclusions/Significance

These results were confirmed by Sanger sequencing. In conclusion, benchtop next-generation sequencing technology can be used to detect mutations in monogenic diseases and can detect both point mutations and indels with high sensitivity, fidelity and throughput at a lower cost than conventional methods in clinical applications.     

Association of Renin Binding Protein (RnBP) Gene Polymorphisms with Essential Hypertension in the Hani Minority of Southwestern China

Essential hypertension is one of the most common multifactorial diseases,affecting 20％-30％ of the human population (Ibrahim and Damasceno,2012).Based on the results of twin studies,adoption studies and statistical analyses of blood pressure (BP) across various pedigrees,it has been estimated that 30％-50％ of the variability in blood pressure among the general population is genetically determined (Garcia et al.,2003).Although the genetic mechanisms of essential hypertension have not been studied well,investigations for the genes that constitute the renin-angiotensin system (RAS) appear to be particularly promising,since this system plays a central role in the regulation of blood pressure (Ferrario,2010).     

美国有机农业发展及对我国的启示

美国有机农业起步较早、发展迅速,已经成为全球有机农产品第一大消费市场,分析美国有机农业起源、发展、标识管理和财政支持对发展我国有机农业、提高我国农产品质量安全具有重要借鉴意义。     

基孔肯雅病毒非结构蛋白基因合成中多位点缺失突变的校正方法

目的:建立一种PCR方法,以快速校正基孔肯雅病毒非结构蛋白基因合成过程中发生的多位点缺失突变。方法:用PCR方法合成基孔肯雅病毒非结构蛋白基因;对测序的克隆进行序列比对,分析不同克隆上缺失突变发生的位置,以保守区域互相重叠的寡核苷酸为上下游引物、以该区域测序正确的克隆为模板进行PCR扩增,得到所需片段,再将这些片段用PCR方法进一步组装成完整的基因序列并进行测序。结果:测序结果表明,经过2次PCR扩增,校正了基孔肯雅病毒非结构蛋白基因合成过程中发生的5个位点缺失突变。结论:得到序列正确的基孔肯雅病毒非结构蛋白基因。在进行基因合成过程中如发生多位点缺失突变,可利用该方法同时对以上突变进行校正,无须再合成引物,降低了实验操作难度,并提高了实验效率。     

Glucose biosensor based on titanium dioxide-multiwall carbon nanotubes-chitosan composite and functionalized gold nanoparticles

In this paper, a new glucose biosensor was prepared. At first, Prussian blue (PB) was electrodeposited on a glassy carbon electrode (GCE) modified by titanium dioxide-multiwall carbon nanotubes-chitosan (TiO₂-MWNTs-CS) composite, and then gold nanoparticles functionalized by poly(diallyldimethylammonium chloride) (PDDA-Au) were adsorbed on the PB film. Finally, the negatively charged glucose oxidase (GOD) was self-assembled on to the positively charged PDDA-Au. The electrochemical performances of the modified electrodes had been studied by cyclic voltammetry (CV) and amperometric methods, respectively. In addition, the stepwise fabrication process of the as-prepared biosensor was characterized by scanning electron microscopy. PDDA-Au nanoparticles were characterized by ultraviolet–vis absorption spectroscopy and transmission electron microscopy. Under the optimal conditions, the as-prepared biosensor exhibited a good response performance to glucose with a linear range from 6 μM to 1.2 mM with a detection limit of 0.1 μM glucose (S/N = 3). In addition, this work indicated that TiO₂-MWNTs-CS composite and PDDA-Au nanoparticles held great potential for constructing biosensors.     

利用移码突变和终止密码子提高下游目的基因的表达水平

目的:在基因结构复杂的慢病毒载体插入报告基因并提高目的基因表达量。方法:慢病毒载体上有复杂的基因排列,为了不影响慢病毒载体的活性,必须尽量保留原有的基因,替换不必要的基因。首先将报告基因萤光素酶插入慢病毒载体替换基因Env后,结果检测不到报告基因的表达。为了提高报告基因的表达水平,将报告基因的读码框向后移动一个碱基,同时在其上游增加一个终止密码子,然后检测报告基因的表达水平。结果:通过移动报告基因的读码框同时在上游增加终止密码子,使报告基因的表达水平大大提高。结论:在构建基因表达载体时,通过改变目的基因与上游起始密码子ATG之间的相对位置以及增加终止密码子,可以大幅提高目的基因的表达水平。