基于简并引物同步克隆两个红麻PDIL同源基因cDNA5'-末端序列

探索利用同一套简并引物结合通用引物同步扩增两个红麻PDIL同源基因的cDNA5’-末端序列,以期为同一转录组中两个旁系同源基因cDNA5' RACE的同步扩增提供借鉴.通过红麻HcPDIL5-2a和HcPDIL5-2b cDNA中间片段及3’-末端已知序列的比时,在其完全保守区段设计了一条引物用于两个基因5' RACE的共反转录;在其部分保守区段设计了两条简并引物,并利用其在两个基因的5'RACE扩增时退火温度的差异,结合通用引物巢式PCR同步扩增两个基因的cDNA 5 ’-末端未知序列.在两个基因全长cDNA拼接序列的基础上设计两对特异引物分别扩增它们的cDNA全长序列,测序结果进一步验证了序列拼接和cDNA 5' RACE同步扩增的可靠性.进化分析证实两个基因属于PDIL基因家族成员.     

向日葵离体再生体系的建立

为了建立高效的向日葵离体再生体系,从基因差异、外植体取材、生长素和细胞分裂素浓度、附加物的添加等方面出发,对向日葵愈伤诱导、分化、生根等过程进行了系统优化。结果表明：杂交材料相对于自交材料更容易实现再生;最佳外植体是生长4 d的子叶;最佳愈伤诱导培养基是MS培养基 (MS) +2.0 mg/L 6-苄基腺嘌呤 (6-BA)+0.5 mg/L奈乙酸 (NAA)+1.0 mg/L激动素 (KT),诱导率最高可达100％;最佳分化培养基是MS+0.2 mg/L 6-BA+0.5 mg/L NAA+0.3 mg/L KT+0.3 mg/L硝酸银 (AgNO3)+0.2 g/L活性炭 (AC),芽分化率可达71％;最佳生根培养基是1/2 MS+0.6 mg/L吲哆丁酸 (IBA),生根率最高为77％。方差分析表明,材料基因型、外植体生长时间、激素、AgNO3、AC对向日葵再生呈现显著性影响。     

新疆短命植物小拟南芥耐盐性的初步研究

利用种子萌发及石蜡切片等方法对新疆短命植物小拟南芥(Arabidopsis pumila)在盐胁迫下种子的萌发率及植株生长发育的变化进行了研究,结果显示:(1)盐胁迫对小拟南芥种子萌发具有抑制作用,当去除盐分后,部分种子能够重新萌发.(2)盐分影响幼苗的形态及内部结构,低盐(50 mmol/L)时,幼苗基生叶较大且数量增加;叶中栅栏组织细胞大,排列舒展;根中导管数量明显增多,植株生长健壮而旺盛;而较高盐(150 mmol/L)时,叶片变小增厚,色深绿,底部叶变黄白色,叶中栅栏组织细胞窄小,排列紧密;根中导管数急剧下降,植株生长受到明显抑制.(3)盐胁迫对成株期植株的高度、花序轴长、角果数等性状都有显著影响,但在NaCl浓度达500 mmol/L时,植株仍然存活且有一定的结实率,显示此时的耐盐性比萌发期和幼苗期有显著提高.研究表明,小拟南芥从幼苗至成株期耐盐性逐渐增强,该特征与其对自然生存环境的适应相关.     

Keratinocyte growth factor/fibroblast growth factor-7-regulated cell migration and invasion through activation of NF-kappaB transcription factors

Keratinocyte growth factor (KGF)/fibroblast growth factor-7 (FGF-7) is a paracrine- and epithelium-specific growth factor produced by cells of mesenchymal origin. It acts exclusively through FGF-7 receptor (FGFR2/IIIb), which is expressed predominantly by epithelial cells, but not by fibroblasts, suggesting that it might function as a paracrine mediator of mesenchymal-epithelial interactions. KGF/FGF-7 plays an essential role in the growth of epithelial cells and is frequently overexpressed in cancers of epithelial origin such as pancreatic cancer, switching paracrine stimulation of KGF/FGF-7 to an autocrine loop. Less is known, however, about the signaling pathways by which KGF/FGF-7 regulates the response of epithelial cells. To delineate the signaling pathways activated by KGF/FGF-7 and examine cellular response to KGF/FGF-7 stimulation, we performed functional analysis of KGF/FGF-7 action. In this report, we show that KGF/FGF-7 activated nuclear factor kappaB (NF-kappaB), which in turn induced expression of VEGF, MMP-9, and urokinase-type plasminogen activator and increased migration and invasion of KGF/FGF-7-stimulated human pancreatic ductal epithelial cells. Expression of phosphorylation-defective IkappaBalpha (IkappaBalphaS32A,S36A), which blocked NF-kappaB activation, inhibited KGF/FGF-7-induced gene expression and cell migration and invasion. Our results demonstrate for the first time that KGF/FGF-7 induces NF-kappaB activation and that NF-kappaB plays an essential role in regulation of KGF/FGF-7-inducible gene expression and KGF/FGF-7-initiated cellular responses. Thus, these findings identify one signaling pathway for KGF/FGF-7-regulated cell migration and invasion and suggest that paracrine sources of KGF/FGF-7 are one of the malignancy-contributing factors from tumor stroma.     

Micropropagation of <Emphasis Type="Italic">Penthorum chinense</Emphasis> through axillary bud

This study reports a protocol for successful micropropagation of Penthorum chinense using nodal explants on Murashige and Skoog (MS) medium supplemented with 6-benzyladenine (BA) or kinetin (Kn). The presence of BA promoted a higher rate of shoot multiplication than Kn. Maximum multiple shoot formation was observed in 59.2% of nodal explants cultured on MS medium supplemented with 2.0 mg l⁻¹ BA after 6 wk. After subculture for 4 wk, the maximum number of shoots (6.4) was obtained on a medium with 2.0 mg l⁻¹ BA, but shoots were too short and not suitable for micropropagation. The taller shoots that regenerated in the presence of lower BA concentration (1.0 mg l⁻¹) were selected for root induction study. Most shoots (98.8%) rooted in the presence of 0.5 mg l⁻¹ indole-3-acetic acid after 3 wk, with each shoot forming an average of 10.0 roots. Plantlets were transferred to soil and successfully acclimatized.     

Phosphorylation and ubiquitination of dynamin-related proteins (AtDRP3A/3B) synergically regulate mitochondrial proliferation during mitosis

The balance between mitochondrial fission and fusion is disrupted during mitosis, but the mechanism governing this phenomenon in plant cells remains enigmatic. Here, we used mitochondrial matrix‐localized Kaede protein (mt‐Kaede) to analyze the dynamics of mitochondrial fission in BY‐2 suspension cells. Analysis of the photoactivatable fluorescence of mt‐Kaede suggested that the fission process is dominant during mitosis. This finding was confirmed by an electron microscopic analysis of the size distribution of mitochondria in BY‐2 suspension cells at various stages. Cellular proteins interacting with Myc‐tagged dynamin‐related protein 3A/3B (AtDRP3A and AtDRP3B) were immunoprecipitated with anti‐Myc antibody‐conjugated beads and subsequently identified by microcapillary liquid chromatography–quadrupole time‐of‐flight mass spectrometry (CapLC Q‐TOF) MS/MS. The identified proteins were broadly associated with cytoskeletal (microtubular), phosphorylation, or ubiquitination functions. Mitotic phosphorylation of AtDRP3A/AtDRP3B and mitochondrial fission at metaphase were inhibited by treatment of the cells with a CdkB/cyclin B inhibitor or a serine/threonine protein kinase inhibitor. The fate of AtDRP3A/3B during the cell cycle was followed by time‐lapse imaging of the fluorescence of Dendra2‐tagged AtDRP3A/3B after green‐to‐red photoconversion; this experiment showed that AtDRP3A/3B is partially degraded during interphase. Additionally, we found that microtubules are involved in mitochondrial fission during mitosis, and that mitochondria movement to daughter cell was limited as early as metaphase. Taken together, these findings suggest that mitotic phosphorylation of AtDRP3A/3B promotes mitochondrial fission during plant cell mitosis, and that AtDRP3A/3B is partially degraded at interphase, providing mechanistic insight into the mitochondrial morphological changes associated with cell‐cycle transitions in BY‐2 suspension cells.     

Structural Basis for Dimerization and Catalysis of a Novel Esterase from the GTSAG Motif Subfamily of the Bacterial Hormone-sensitive Lipase Family

Hormone-sensitive lipases (HSLs) are widely distributed in microorganisms, plants, and animals. Microbial HSLs are classified into two subfamilies, an unnamed new subfamily and the GDSAG motif subfamily. Due to the lack of structural information, the detailed catalytic mechanism of the new subfamily is not yet clarified. Based on sequence analysis, we propose to name the new subfamily as the GTSAG motif subfamily. We identified a novel HSL esterase E25, a member of the GTSAG motif subfamily, by functional metagenomic screening, and resolved its structure at 2.05 Å. E25 is mesophilic (optimum temperature at 50 °C), salt-tolerant, slightly alkaline (optimum pH at 8.5) for its activity, and capable of hydrolyzing short chain monoesters (C2–C10). E25 tends to form dimers both in the crystal and in solution. An E25 monomer contains an N-terminal CAP domain, and a classical α/β hydrolase-fold domain. Residues Ser¹⁸⁶, Asp²⁸², and His³¹² comprise the catalytic triad. Structural and mutational analyses indicated that E25 adopts a dimerization pattern distinct from other HSLs. E25 dimer is mainly stabilized by an N-terminal loop intersection from the CAP domains and hydrogen bonds and salt bridges involving seven highly conserved hydrophilic residues from the catalytic domains. Further analysis indicated that E25 also has some catalytic profiles different from other HSLs. Dimerization is essential for E25 to exert its catalytic activity by keeping the accurate orientation of the catalytic Asp²⁸² within the catalytic triad. Our results reveal the structural basis for dimerization and catalysis of an esterase from the GTSAG motif subfamily of the HSL family.