桃ERF转录因子家族生物信息学分析及 芽萌发相关基因筛选

以中油四号油桃(Prunus persica var. nectarina)为研究对象, 利用MEGA 6.0、MEME、GSDS和DNAMAN 6.0等软件对桃ERF家族数据进行生物信息学分析, 鉴定得到102个ERF转录因子家族基因, 并通过构建系统进化树将这102个基因分为10个子家族(I-X)。基因结构分析表明, 有81个基因不含内含子, 20个基因含有1个内含子, 有1个基因与其它成员差异较大, 含有5个内含子。保守元件分析表明, ERF家族包含20个保守元件, 其中Motif 1、Motif 2和Motif 4都属于AP2/ERF结构域, 同一个保守元件主要出现在同一个子家族中, 并且大部分保守元件的功能未知。VIII子家族基因的荧光定量PCR分析表明, 在桃叶芽处于不同的发育状态时, PpeERF068的表达量存在较大差异, 光照培养箱中培养的桃芽在萌发过程中各时期表达量变化趋势进一步表明该基因可能与叶芽萌发有关, 将其命名为PpeEBB1。该研究为进一步揭示PpeEBB1的分子机制奠定了基础, 并为桃树的栽培管理和熟期调控了提供理论指导。     

Detection and Quantification of Fusarium oxysporum f. sp. niveum Race 1 in Plants and Soil by Real-time PCR

Fusarium wilt caused by Fusarium oxysporum f. sp. niveum (Fon) is the most serious soil-borne disease in the world and has become the main limiting factor of watermelon production. Reliable and quick detection and quantification of Fon are essential in the early stages of infection for control of watermelon Fusarium wilt. Traditional detection and identification tests are laborious and cannot efficiently quantify Fon isolates. In this work, a real-time polymerase chain reaction (PCR) assay has been described to accurately identify and quantify Fon in watermelon plants and soil. The FONRT-18 specific primer set which was designed based on identified specific sequence amplified a specific 172 bp band from Fon and no amplification from the other formae speciales of Fusarium oxysporum tested. The detection limits with primers were 1.26 pg/μl genomic DNA of Fon, 0.2 pg/ng total plant DNA in inoculated plant, and 50 conidia/g soil. The PCR assay could also evaluate the relationships between the disease index and Fon DNA quantity in watermelon plants and soil. The assay was further used to estimate the Fon content in soil after disinfection with CaCN₂. The real-time PCR method is rapid, accurate and reliable for monitoring and quantification analysis of Fon in watermelon plants and soil. It can be applied to the study of disease diagnosis, plant-pathogen interactions, and effective management.     

The affinity of human RANK binding to its ligand RANKL

Receptor activator of nuclear factor-kappa B (RANK) and its ligand, RANKL play critical roles in bone re-modeling, immune function, vascular disease and mammary gland development. To study the interaction of RANK and RANKL, we have expressed both extracellular domain of RANK and ectodomain of RANKL using Escherichia coli expression system. RANK was expressed as an inclusion body first which properly refolded later, while RANKL was initially produced as a GST fusion protein, after which the GST was removed by enzyme digestion. Soluble RANK existed as a monomer while RANKL was seen as a trimer in solution, demonstrated by gel filtration chromatography and cross-linking experiment. The recombinant RANK and RANKL could bind to each other and the binding affinity of RANKL for RANK was measured with surface plasmon resonance technology and K_D value is about 1.09 × 10⁻¹⁰ M.     

Removal of selectable marker gene from fibroblast cells in transgenic cloned cattle by transient expression of Cre recombinase and subsequent effects on recloned embryo development

Introduction of selectable marker genes to transgenic animals could create an inconvenience to further research and may exaggerate public concerns regarding biological safety. The objective of the current study was to excise loxP flanked neo^R in transgenic cloned cattle by transient expression of Cre recombinase. Green fluorescent protein gene (GFP) was incorporated to monitor Cre expression; therefore, Cre-expressed cells could be selected indirectly by fluorescence-activated cell sorting (FACS). The neo^R was removed and Cre expressed transiently in GFP-positive colonies; excision of neo^R was confirmed by single-blastocyst PCR in recloned blastocysts, with neo^R-free fibroblast cells as donors. There was no difference (P > 0.05) in rates of cleavage (76.0% vs. 68.8%) or blastocyst formation (56.6% vs. 52.9%) between recloned embryos with neo^R-free or neo^R-included donors. The differential staining of recloned blastocysts were similar (P >0.05) in terms of total cell number (124 vs. 122) and the ratio of ICM (Inner Cell Mass) to the total cell number (38.1% vs. 38.2%). Furthermore, pregnancy and calving rates were not different (P > 0.05) from those of the control. In conclusion, we successfully excised neo^R from transgenic cloned cattle; the manipulation did not affect the developmental competence of recloned preimplantation embryos. This approach should benefit bioreactor and transgenic research in livestock.     

Spindlin1, a novel nuclear protein with a role in the transformation of NIH3T3 cells

spindlin1, a novel human gene recently isolated by our laboratory, is highly homologous to mouse spindlin gene. In this study, we cloned cDNA full-length of this novel gene and send it to GenBank database as spindlin1 (Homo sapiens spindlin1) with Accession No. AF317228. In order to investigate the function of spindlin1, we studied further the subcellular localization of Spindlin1 protein and the effects of spindlin1 overexpression in NIH3T3 cells. The results showed that the fusion protein pEGFP-N1-spindlin1 was located in the nucleus and the C-terminal is correlated with nuclear localization of Spindlin1 protein. NIH3T3 cells which could stably express spindlin1 as a result of RT-PCR analysis compared with the control cells displayed a complete morphological change; made cell growth faster; and increased the percentage of cells in G2/M and S phase. Furthermore, overexpressed spindlin1 cells formed colonies in soft agar in vitro and formed tumors in nude mice. Our findings provide direct evidence that spindlin1 gene may contribute to tumorigenesis.     

Combined analysis of the association between p73 G4C14-to-A4T14 polymorphisms and cancer risk

P73 is a structural and functional homologue of p53, and plays an important role in regulating cell cycle and apoptosis. A potentially functional polymorphism (designated as p73 G4C14-to-A4T14) has been identified in a region in exon 2 of the p73 gene, which may theoretically form a stem-loop structure and thereby affect p73 expression. Several investigations have reported the correlation between p73 G4C14-to-A4T14 polymorphism and cancer risk. However, the results are inconclusive. To further assess the association between p73 polymorphism and cancer risk, we performed meta-analysis of the data sets obtained from 26 individual studies involving 8,148 cancer patients and 8,150 controls. The association between p73 G4C14-to-A4T14 polymorphism and cancer risk was determined by crude odd ratios (OR) with 95% CI (confidential interval). AT-allele carriers were found to have a significantly increased risk of cervical cancer (AT/GC vs. GC/GC, OR = 1.63, 95% CI = 1.14–2.33; AT/AT + AT/GC vs. GC/GC, OR = 1.49, 95% CI = 1.05–2.10), colorectal cancer (AT/AT vs. AT/GC + GC/GC, OR = 1.98, 95% CI = 1.25–3.12), head and neck cancer (AT/AT + AT/GC vs. GC/GC, OR = 1.44, 95% CI = 1.06–1.96) and other cancers (AT/AT vs. GC/GC, OR = 1.78, 95% CI = 1.24–2.57; AT/AT vs. AT/GC + GC/GC, OR = 1.80, 95% CI = 1.26–2.56). In the stratified analysis of ethnicity, a significantly elevated cancer risk was found in Caucasians (AT/AT + AT/GC vs. GC/GC, OR = 1.18, 95% CI = 1.08–1.30; allele AT vs. allele GC, OR = 1.15, 95% CI = 1.06–1.24). No significant association of p73 polymorphism with the cancer risk of smoking was detected by stratified analysis by smoking status. Together, our data suggest that the p73 G4C14-to-A4T14 may be a risk factor of cancer especially in Caucasians.     

IGF-1 Reduces BACE-1 Expression in PC12 Cells via Activation of PI3-K/Akt and MAPK/ERK1/2 Signaling Pathways

Insulin-like growth factor 1 (IGF-1) stimulates α-secretase processing of amyloid precursor protein (APP) and decreases Aβ production. Little is known about the relationship between IGF-1 and β-site amyloid precursor protein cleaving enzyme 1 (BACE-1), the protease essential for the production of β-amyloid peptides (Aβ). Here, we investigated the effect of IGF-1 on BACE-1 in PC12 cells. Quantitative polymerase chain reaction analysis and western blot showed that treatment of cells with IGF-1 significantly decreased the levels of BACE-1 mRNA and protein. Furthermore, IGF-1 increased the phosphorylation of Akt and ERK1/2. The presence of the phosphatidylinositol 3-kinase (PI3-K) inhibitor LY294002 and the mitogen-activated protein kinase kinases (MEK) inhibitor PD98059 blocked the effect of IGF-1 on BACE-1. Our data indicated that IGF-1-induced reduction of BACE-1 might involve the PI3-K/Akt and MAPK/ERK1/2 signaling pathways.