One-carbon compounds transport studies in the obligate methylotroph

Cells of obligate methylotrophic Gram-negative bacterium Methylobacillus flagellatum KT which can only grow on methanol and methylamine media possess three different carriers mediating uptake of methylamin depending on growth conditions. All three uptake systems are energy-dependent, the methylamine uptake was inhibited by oxidative phosphorylation uncoupler and respiratory inhibitors. The first active transport system for methanol in the cells of obligate methylotroph was also demonstrated. The parameters of this system were measured, their dependence on energy, presence of respiratory inhibitors and uncoupler was shown.Abbreviations CCCP Carbonyl cyanide p-(trichloromethoxy)-phenylhydrazone - DCCD N,N-dicyclohexyl-carbodiimide     

Comparative study on the replication of HCV1b genome between wild-type and cell culture-adaptive mutant in regard to sensitivities against anti-HCV drugs

The replicon system, which mimics viral genome replication in culture cells, has been widely used to analyze the genome replication of the hepatitis C virus (HCV). However, most HCV genomes used in the system include adaptive mutations (AMs) that are vital for replication in culture cells despite the nonexistence of such mutations in the genome of wild-type (WT) HCV in patients. In order to study the genome replications of WT HCV, new HCV subgenomic replicon (SGR) systems were established using Huh-7.5-derived cells producing Sec14-like protein 2 constitutively and SGR of KT9 (one of the HCV genotype 1b clones) with WT genome (SGR KT9WT) in this study. The replication efficiency and sensitivities of SGR KT9WT to anti-HCV drugs in the cloned cells permanently bearing replicon RNA, HS55-4 cells, were similar to those of reports using SGR, including AM. The SGR transient transfection system using SGR KT9WT and SGR KT9AM encoding secreted Nano-luciferase and HS55-4C cells established by the elimination of SGR KT9 RNA from HS55-4 cells, however, showed that the replication efficiency of SGR KT9WT was much lower than that of SGR KT9AM under a same condition. Furthermore, the sensitivities of SGR KT9WT to almost all tested anti-HCV reagents, except the inhibitor of miR-122, a cellular factor important for HCV replication, were quite low compared with SGR KT9AM. These results suggested that the new replicon systems might not only provide information about precise responses against new anti-HCV drugs but also reveal novel molecular mechanisms supporting negligent proliferation of HCV.     

KT和NAA对黄芪带芽茎段增殖、愈伤诱导和生根的影响

通过建立组织培养快繁体系,探讨KT和NAA对膜荚黄芪茎段诱导生成新生茎、愈伤组织及生根的影响。KT/NAA值为4时,对茎的诱导作用明显,获得最多的茎平均数(11.7条)。NAA促进愈伤组织形成,在1 mg/L NAA作用下最大的平均直径为1.4 cm,多为白色。根生于白色愈伤组织,并受NAA促进、KT抑制。1 mg/L NAA对根数、根长和次级根数均有促进作用,但对根粗有抑制作用。     

解除休眠处理对凤丹种子萌发和幼苗生长的影响

凤丹种子具有很深的休眠特性。本实验以AgNO₃、GA₃、KT、TDZ和4℃低温处理凤丹种子,探讨解除其上胚轴休眠的途径。结果表明,AgNO₃、GA₃和低温(4℃)处理能加快凤丹种子萌发并提高发芽率,且不同程度地改善幼苗的鲜重、株高、根系等生长指标。     

Application of free-flow electrophoresis/2-dimentional gel electrophoresis for fractionation and characterization of native proteome of Pseudomonas putida KT2440

Free Flow Electrophoresis (FFE) is a liquid-based isoelectric focusing method. Unlike conventional in-gel fractionation of proteins, FFE can resolve proteins in their native forms and fractionation of subcellular compartments of the cell is also possible. To test the efficacy of the FFE method, the native cytosol proteome of a bacterium, Pseudomonas putida KT2440 was fractionated by FFE and the spectrum of protein elutes was characterized in association with 2-dimentional gel electrophoresis (2-DE). Major native proteins of P. putida KT2440 were eluted in the range of pH 4.8 approximately 6.0 in FFE, whereas the denatured proteome of P. putida KT2440 was widely distributed in the rage of pH 4 approximately 10 in the 2-DE analysis. In addition, one of the three FFE major fractions, which was eluted at pH 5.0, was further analyzed using 2-DE/MS-MS. Then, the pH range of identified proteins eluted in 2-DE/MS-MS was 4.72 approximately 5.89, indicating that observed pi values of native cytosolic proteomes in FFE were narrower than those of denatured cytosolic proteome. These results suggest that FFE fractionation and 2-DE/MS analysis may be useful tools for characterization of native proteomes of P. putida KT2440 and comparative analysis between denatured and native proteomes.     

大豆KUP/HAK/KT钾转运体基因家族的鉴定与表达分析

该研究基于已公布的大豆基因组序列信息,对大豆KUP/HAK/KT钾转运体基因家族进行了全基因组鉴定,并对该家族成员的基因特征、蛋白结构、染色体定位、基因复制和表达模式等进行了全面分析,为进一步了解该家族基因的功能及培育钾高效大豆品种提供理论支撑。结果表明:(1)在大豆基因组中共鉴定30个KUP/HAK/KT基因(简写为GmHAK01~GmHAK30),这些基因分布在大豆的15条染色体上,串联复制和片段复制可能导致了GmHAKs基因在大豆基因组中的扩增。(2)大豆GmHAKs蛋白间序列一致性很高,均具有12~14个跨膜区,且都定位于质膜上。(3)进化分析表明大豆GmHAKs可聚为4个进化簇ClusterⅠ~Ⅳ,其中ClusterⅡ的成员数目最多(16个),ClusterⅣ的成员数目最少(1个)。(4)所有GmHAKs基因均包含内含子和外显子,其内含子数目在7~9个之间,且同一亚家族的GmHAKs基因大部分具有相似的内含子-外显子分布模式。(5)表达模式分析表明,大豆GmHAKs的表达大致可分为两类:一类是一些组织特异性表达的基因,包括了ClusterⅠ和ClusterⅣ的全部成员,ClusterⅡ的部分成员,他们在根(GmHAK30和GmHAK04)、花(GmHAK03和GmHAK15)、荚(GmHAK10)或种子(GmHAK25)中表达量很高;另外一类是一些非组织特异性表达的基因,包括了ClusterⅢ的全部成员和ClusterⅡ的部分成员,这些基因(GmHAK05、GmHAK17和GmHAK28等)在所有被检测的组织中均有较高的表达;KUP/HAK/KT家族基因表达模式在不同进化簇的差异化结果表明,其在进化过程可能受到了选择的作用。以上研究结果为今后研究KUP/HAK/KT家族基因功能及定向改良大豆的钾吸收物性提供了重要的基因信息,也为大豆钾高效品种的选育提供了理论基础。