Towards genomic and proteomic studies of protein phosphorylation in plant-pathogen interactions

Phosphorylation is an effective method of post-translational protein modification but understanding its significance is hindered by its biological complexity. Many protein kinases and phosphatases have been identified that connect signal perception mechanisms to plant defence responses. Recent studies of mitogen-activated protein kinases, calcium-dependent protein kinases and other kinases and phosphatases have revealed some important mechanisms, but have also raised new questions. The regulation of any phosphorylation pathway is complex and dynamic. There are many protein kinases and phosphatases in the plant genome, which makes it hard to delineate the phosphorylation machinery fully. Genomics and proteomics have already identified new components and will continue to influence the study of phosphorylation profoundly in plant-pathogen interactions.     

小鼠胚胎干细胞定向分化为神经干细胞过程中microRNA的变化

目的用生物芯片技术分析胚胎干细胞定向分化为神经干细胞过程中microRNA(miRNA)的表达变化,筛选调控的分化的miRNA,研究分化调控机制。方法胚胎干细胞在含LIF培养基中培养3d后,采用经典5步培养方法定向诱导向神经干细胞分化,采用nestin作为神经干细胞标记进行鉴定,送检胚胎干细胞及神经干细胞,提取总RNA以及小分子RNA,经荧光标记后与miRNA基因芯片杂交,获得胚胎干细胞诱导前后miRNA表达谱。结果1)胚胎干细胞在含LIF培养过程中保持未分化状态,Oct-4、碱性磷酸酶表达阳性;2)经典五步法诱导胚胎干细胞定向分化为神经干细胞,nestin阳性细胞为85%;3)通过基因微阵列分析,有90个miRNA的改变显著,其中68个表达上调,22个表达下调。结论miRNA可能对胚胎干细胞定向分化为神经干细胞过程起到关键作用。     

云芝子实体多糖(CVP)化学结构的研究III.

通过化学、IR和UV光谱等方法发现云芝子实体的热水提取物 CVP 是由多糖、核酸和蛋白质等组成的混合物。通过柱层析方法从 CVP 中纯化出一个多糖组份 B-2-3,经完全水解证实组成它的单糖残基主要为Glc。用 HPLC 方法测得其分子量为 5.01×105。通过对甲基化及 1H 和 13C NMR 数据的分析发现 B-2-3 为一个以β-D-1, 4-Glc、β-D-1, 3-Glc 和β-D-1, 6-Glc 残基构成主链,同时在β-D-1, 3, 6-Glc 及β-D-1, 4, 6-Glc 处产生分枝的多糖。     

Analysis of trehalose-6-phosphate synthase (TPS) gene family suggests the formation of TPS complexes in rice

Trehalose-6-phosphate (T6P), an intermediate in the trehalose biosynthesis pathway, is emerging as an important regulator of plant metabolism and development. T6P levels are potentially modulated by a group of trehalose-6-phosphate synthase (TPS) and trehalose-6-phosphate phosphatase (TPP) homologues. In this study, we have isolated 11 TPS genes encoding proteins with both TPS and TPP domains, from rice. Functional complement assays performed in yeast tps1 and tps2 mutants, revealed that only OsTPS1 encodes an active TPS enzyme and no OsTPS protein possesses TPP activity. By using a yeast two-hybrid analysis, a complicated interaction network occurred among OsTPS proteins, and the TPS domain might be essential for this interaction to occur. The interaction between OsTPS1 and OsTPS8 in vivo was confirmed by bimolecular fluorescence complementation and coimmunoprecipitation assays. Furthermore, our gel filtration assay showed that there may exist two forms of OsTPS1 (OsTPS1a and OsTPS1b) with different elution profiles in rice. OsTPS1b was particularly cofractionated with OsTPS5 and OsTPS8 in the 360 kDa complex, while OsTPS1a was predominantly incorporated into the complexes larger than 360 kDa. Collectively, these results suggest that OsTPS family members may form trehalose-6-phosphate synthase complexes and therefore potentially modify T6P levels to regulate plant development.     

Change in microbial communities in acetate- and glucose-fed microbial fuel cells in the presence of light

Power densities produced by microbial fuel cells (MFCs) in natural systems are changed by exposure to light through the enrichment of photosynthetic microorganisms. When MFCs with brush anodes were exposed to light (4000 lx), power densities increased by 8–10% for glucose-fed reactors, and 34% for acetate-fed reactors. Denaturing gradient gel electrophoresis (DGGE) profiles based on the 16S rRNA gene showed that exposure to high light levels changed the microbial communities on the anodes. Based on 16S rRNA gene clone libraries of light-exposed systems the anode communities using glucose were also significantly different than those fed acetate. Dominant bacteria that are known exoelectrogens were identified in the anode biofilm, including a purple nonsulfur (PNS) photosynthetic bacterium, Rhodopseudomonas palustris, and a dissimilatory iron-reducing bacterium, Geobacter sulfurreducens. Pure culture tests confirmed that PNS photosynthetic bacteria increased power production when exposed to high light intensities (4000 lx). These results demonstrate that power production and community composition are affected by light conditions as well as electron donors in single-chamber air-cathode MFCs.     

外源α-萘乙酸对花期干旱大豆碳代谢的影响

以耐旱性大豆品种晋豆21和干旱敏感性大豆品种徐豆22为试验材料,通过盆栽试验,研究α-萘乙酸(NAA)对花期干旱大豆碳代谢的影响.结果表明: 干旱胁迫下,与徐豆22相比,晋豆21净光合速率(P_n)下降幅度较小,光呼吸速率(P_r)和叶片可溶性糖含量增加幅度较小,而叶片蔗糖磷酸合成酶(SPS)、蔗糖合成酶(SS)(合成方向)活性、根系蔗糖含量增加幅度较大.NAA处理提高了干旱胁迫下P_n,并降低了P_r,进而明显缓解了干旱胁迫对大豆植株的生长抑制;降低了叶片淀粉分解酶、酸性转化酶(AI)和SS(分解方向)活性,从而抑制了干旱胁迫诱导的可溶性糖积累;NAA处理也能增加干旱胁迫下叶片SPS、SS(合成方向)活性、根系蔗糖含量、根冠比,表明NAA处理促进了叶片中蔗糖向根系的转运.总之,在干旱胁迫下,外源NAA能够通过调控碳代谢增强大豆植株对干旱胁迫的耐受性.     

乏情和发情初产母猪下丘脑–垂体–卵巢轴中lincRNAs表达谱比较分析

初产母猪断奶后能否正常发情对养猪生产影响重大,也是初产母猪被淘汰的主要原因。本研究以乏情和发情初产母猪为研究对象,首次利用RNA-seq技术对其下丘脑-垂体-卵巢轴中的基因间长链非编码RNAs(long intergenic noncoding RNAs,lincRNAs)进行筛选比较,得到lincRNAs的表达图谱,并对其特征和功能进行了初步分析。结果显示,在乏情和发情初产母猪下丘脑–垂体–卵巢轴中鉴定得到3519个lincRNAs,以发情组为对照共有17个lincRNAs存在差异表达,其中12个表达上调,5个表达下调(FC≥2,P<0.05)。选择4个差异表达的lincRNAs经qRT-PCR验证,其表达水平与测序结果基本一致。对这17个差异表达的lincRNAs进行GO分析、KEGG通路分析及lincRNA-mRNA共表达网络分析,发现这些lincRNAs主要与猪卵母细胞减数分裂成熟、卵巢细胞分化及颗粒细胞凋亡等生殖活动相关。本研究结果丰富了猪lincRNAs数据资源,为进一步深入研究初产母猪的生殖机能提供了理论依据。     

锡金海棠子叶再生体系的建立

锡金海[Malussikkimensis（Wenz．）Koehne]是我国稀有植物和优良苹果砧木种质资源。本研究探讨了影响锡金海棠离体再生的条件,建立了锡金海棠子叶高效离体再生体系。结果表明,子叶再生体系的最佳诱导培养基SC＋0．1mg·L-1 NAA＋2．0mg·L-1 TDZ,最佳分化培养基SC＋0．1mg·L-1 NAA＋1．5mg·L-1 TDZ,最佳继代增殖培养基SC＋2．0mg·L-1 6-BA＋0．2mg·L-1 NAA,最佳生根培养基为1／2MS＋I．0mg·L-1 IBA。炼苗后,移入珍珠岩、花生壳与草炭（1：1：1）的基质中,20d移栽成活率高达90％。     

Enzyme-assisted extraction of flavonoids from Ginkgo biloba leaves: improvement effect of flavonol transglycosylation catalyzed by Penicillium decumbens cellulase

We report a novel enzyme-involved approach to improve the extraction of flavonoids from Ginkgo biloba, in which the enzyme is employed not only for cell wall degradation, but also for increasing the solubility of target compounds in the ethanol-water extractant. Penicillium decumbens cellulase, a commercial cell wall-degrading enzyme with high transglycosylation activity, was found to offer far better performance in the extraction than Trichoderma reesei cellulase and Aspergillus niger pectinase under the presence of maltose as the glycosyl donor. TLC, HPLC and MS analysis indicated that P. decumbens cellulase could transglycosylate flavonol aglycones into more polar glucosides, the higher solubility of which led to improved extraction. The influence of glycosyl donor, pH, solvent and temperature on the enzymatic transglycosylation was investigated. For three predominant flavonoids in G. biloba, the transglycosylation showed similar optimal conditions, which were therefore used for the enzyme-assisted extraction. The extraction yield turned to be 28.3mg/g of dw, 31% higher than that under the pre-optimized conditions, and 102% higher than that under the conditions without enzymes. The utilization of enzymatic bifunctionality described here, naming enzymatic modification of target compounds and facilitation of cell wall degradation, provides a novel approach for the extraction of natural compounds from plants.     

Application of a novel thermostable NAD(P)H oxidase from hyperthermophilic archaeon for the regeneration of both NAD⁺ and NADP⁺

A novel thermostable NAD(P)H oxidase from the hyperthermophilic archaeon Thermococcus kodakarensis KOD1 (TkNOX) catalyzes oxidation of NADH and NADPH with oxygen from atmospheric air as an electron acceptor. Although the optimal temperature of TkNOX is >90°C, it also shows activity at 30°C. This enzyme was used for the regeneration of both NADP(+) and NAD(+) in alcohol dehydrogenase (ADH)-catalyzed enantioselective oxidation of racemic 1-phenylethanol. NADP(+) regeneration at 30°C was performed by TkNOX coupled with (R)-specific ADH from Lactobacillus kefir, resulting in successful acquisition of optically pure (S)-1-phenylethanol. The use of TkNOX with moderately thermostable (S)-specific ADH from Rhodococcus erythropolis enabled us to operate the enantioselective bioconversion accompanying NAD(+) regeneration at high temperatures. Optically pure (R)-1-phenylethanol was successfully obtained by this system after a shorter reaction time at 45-60°C than that at 30°C, demonstrating an advantage of the combination of thermostable enzymes. The ability of TkNOX to oxidize both NADH and NADPH with remarkable thermostability renders this enzyme a versatile tool for regeneration of the oxidized nicotinamide cofactors without the need for extra substrates other than dissolved oxygen from air.