Role of Daxx in inhibiting megakaryocytic differentiation of K562 cells

AIM: To examine the effects of death domain-associated protein (Daxx) overexpression on the viability and megakaryocytic differentiation of K562 cells. METHODS: Daxx overexpression in the K562 cells was established. The expression of Daxx was detected by fluorescence microscopy, fluorescence quantitative real-time PCR and Western blot after transfection. CCK-8 assay was used to detect the cell viability after overexpression of Daxx. The expression of CD41 and CD61 in phorbol 12-myristate 13-acetate (PMA) induced K562 cells was detected by flow cytometry. The protein levels of Daxx and p-ERK were determined by Western blot. Nitroblue tetrazolium (NBT)-reducing test was used to assess leukemia cell differentiation in Daxx-overexpressing K562 cells and control cells. The expression of CD41 and CD61 induced by PMA in Daxx-overexpressing K562 cells was analyzed by flow cytometry. The protein levels of Daxx and p-ERK were also examined by Western blot. RESULTS: The stable overexpression of Daxx in the K562 cells was established. The viability was reduced in Daxx-overexpressing K562 cells. The expression of CD41 and CD61 was significantly increased after PMA induction in the K562 cells (P < 0.01). The protein expression of Daxx was reduced, but the protein level of p-ERK was increased. The expression of CD41 and CD61 was reduced after PMA induction in Daxx-overexpressing K562 cells (P < 0.01). The protein level of p-ERK was also reduced. CONCLUSION: Daxx overexpression inhibits the growth, megakaryocytic differentiation and production of p-ERK in the K562 cells.     

萝卜RsCYP86MF 基因cDNA全序列克隆及结构特征分析

 本研究通过设计保守的基因特异引物, 运用SMART RACE RT - PCR技术从‘圆白’萝卜中获得一个细胞色素P450基因, 命名为RsCYP86M F, 其cDNA全长1818 bp, 含有1581 bp的完整开放阅读框, 编码526个氨基酸, 该基因编码的蛋白序列与白菜的细胞色素P450基因编码的氨基酸序列同源性高达90% , 且有很高的功能区段保守性; 对其可能编码的蛋白质二级结构进行预测发现, 该蛋白质具有细胞色素P450典型的保守结构域FNAGPRLCIG, 及N - 糖基化位点等结构域。     

Temporal variation of characteristic scales in urban landscapes: an insight into the evolving internal structures of China’s two largest cities

Urbanization has induced profound landscape changes. While the spatiotemporal patterns of urban landscapes have been extensively studied, the manner by which the internal structures of already urbanized areas change remains little understood. Characteristic scales are an important measure of landscape structure, and they represent the typical spatial extents of landscape elements in hierarchies. In this study, we quantified temporal variations of the characteristic scales in the central urban landscapes of Beijing and Shanghai over an 18?year period. Using transect data from Landsat images, characteristic scales were identified through wavelet analysis and then classified into several discrete domains using the k-means clustering method. These characteristic scale domains appeared to correspond with the typical extents of the blocks and block clusters in the study areas. Results showed that the number of the characteristic scale domains changed within a small range of 3?C5 while the mean values of the characteristic scales within the domains showed substantial temporal variation. Larger characteristic scales were more variable than smaller ones in both cities. Distinguishing relative change rates of building forms, land use and street layout of urban landscapes allowed us to interpret these differences. The street layout of urban landscapes usually reacts slowly to the force of change, acting as the skeleton of the urban landscape. As a result, block sizes can remain relatively stable and corresponding characteristic scales present inheritance features. Land use and building forms are more susceptible to changes. Block clusters with flexible extents could result in significant variation of characteristic scales.     

辣椒CaCOI1 基因的克隆、表达及其序列分析

 利用RT-PCR技术从辣椒中克隆到基因CaCOI1,该基因编码的蛋白质由603个氨基酸残基组成,蛋白质的分子量为68.35 kD,等电点是6.32。在蛋白质N–端有1个F-box结构域,C–端有6个富含亮氨酸结构域。二级结构分析表明,CaCOI1蛋白分子中,α–螺旋、β–折叠、β–转角和不规则卷曲分别为51.41%、13.76%、5.80%和29.03%。CaCOI1蛋白的平均亲水系数为–0.143,为亲水蛋白。蛋白质序列比对和进化树分析表明,CaCOI1与番茄LeCOI1蛋白的一致性最高（94%）,进化距离最近。实时定量RT-PCR分析表明,CaCOI1在辣椒的根、茎、幼叶、成熟叶、花、青熟期果实、成熟红果等不同生长发育时期的组织中都能表达,在花中表达水平最高,是其他组织的2.8 ~ 5.4倍,表明CaCOI1在花的发育过程中起重要作用。     

南瓜 NBS类抗病基因同源序列的克隆与分析

依据已克隆植物抗病基因的保守氨基酸结构域合成相应简并引物,通过PCR扩增从南瓜基因组DNA中分离了8条南瓜抗病基因同源序列,通过对其编码的氨基酸序列分析表明这些RGAs均含有P-loop及GLPL等植物抗病基因中的保守结构域.经BLAST分析表明,分离的南瓜RGAs与已报道的甜瓜等植物的RGAs有较高的同源性.南瓜RGA的分离将为进一步从南瓜中分离功能性抗病基因打下基础,也为研究南瓜种质资源的起源与进化提供借鉴.     

百合品种抗病基因同源序列分析及抗枯萎病的鉴定

 采用鳞片接种法对36个百合品种及5个野生种进行枯萎病（Fusarium oxysporum f. sp. lilii）接种鉴定,筛选抗性优异资源。结果表明：百合品种及野生种中蕴藏丰富的抗枯萎病种质资源。利用RGA-PCR方法对百合遗传多样性进行分析,10对RGA引物在41个品种及野生种中共扩增条带159条,其中多态带156条,占总带数的98.1%。以相似系数0.71聚类,41个百合品种及野生种划分为7类。抗性表型与RGA相似性聚类结果表明,利用品种的抗性表型聚类,趋于抗病性相近的聚为一类;利用RGA相似性聚类,趋于遗传背景相近的聚为一类。百合品种RGA的DNA指纹多态性与枯萎病抗性遗传表型多样性无明显的对应关系。对亲本材料进行抗性鉴定和抗病基因同源序列多态性分析,能更准确地反映亲本的遗传背景,为百合枯萎病抗性基因鉴定及品种培育提供一定的理论依据。     

苹果MdGLRs家族基因生物信息学鉴定和表达分析

 利用生物信息学策略对苹果MdGLRs家族基因编码蛋白的氨基酸序列的基本特征、二级结构、跨膜区域、亲疏水性、基因亚细胞定位及保守结构域进行了预测和分析,与拟南芥AtGLRs家族的20个基因进行了氨基酸序列比对和进化树分析,并对这些基因在不同营养生长器官中进行了表达分析。结果表明,苹果MdGLRs家族包含32个基因,分为3个亚族,大部分基因编码800个氨基酸以上,多含有3个跨膜区域,二级结构主要以α–螺旋、无规则卷曲、折叠延伸链为主;亚细胞定位预测发现,MdGLRs蛋白主要定位在内质网和质膜上;MdGLRs蛋白的保守结构域是S1-M1-M2-M3-S2-M4;大多数基因在根、茎、叶中普遍都有表达,在叶中的表达量最高。     

Spatial and temporal dynamics of habitat availability and stability for a critically endangered arboreal marsupial: implications for conservation planning in a fire-prone landscape

Landscape Ecology - Effective conservation planning for species depends on vegetation models that can capture the dynamics of habitat elements across both spatial and temporal domains....     

Differential expression of the ascorbate oxidase multigene family of Camellia sinensis in response to stress

Apoplastic ascorbate oxidase (AO) plays a major role in cell growth. Although AO genes have been studied in depth, some articles have mistakenly identified AO homologues as AO genes. Overall, the divergence between AO genes and AO homologues has not been explored. Meanwhile, there is little information concerning AO and the AO homologue with respect to Camellia sinensis. In the present study, one CsAO homologue and three CsAOs were confirmed by RT-PCR amplification, cloning and sequencing. Multicopper oxidase type 1 (PF00394), type 2 (PF07731) or type 3 (PF07732) domains and one transmembrane helix were the key domains for each member of the cupredoxin family. The CsAOs with their counterparts from seven dicotyledonous plants and three monocotyledonous plants were used to build phylogenetic tree and compare the deduced polypeptides. CsAO may be strongly expressed in the stretch expanded tissues, including bud and root. The abiotic stress-induced expression pattern of the CsAO homologue (CsAO2) is similar to those of CsAO1, CsAO3 and CsAO4. A new and very large group of AO homologues, which may function as AO genes, was present in both dicotyledonous and monocotyledonous plants. Our study may help in identifying stress-responsive AO genes of plants.     

Spatial and temporal heterogeneity of forest site productivity drivers: a case study within the eastern boreal forests of Canada

Forest productivity is driven by a suite of direct climatic and non-climatic factors that are transient or permanent. The kind of productivity driver and the nature of their effects vary by species, and scale dependencies potentially complicate these relationships. This study explored productivity-driver relations in eastern Boreal Canada and determined spatial effects in productivity control when expressed with stand dominant height at a reference age (site index). Data from 4,217 temporary sample plots obtained from boreal mixedwood and conifer bioclimatic domains, and with varied species composition, were used in this study. A single-level global model that assumes equal sensitivities across spatial scales was calibrated and compared with three alternative models reflecting different hypotheses on possible spatial heterogeneities. Alternative models were calibrated by plot-level soil deposit types (microscale), landscape dominant deposits (mesoscale) and bioclimatic domains (macroscale). A marked difference between the global and alternative models was observed, suggesting that a single global model does not sufficiently reflect existing heterogeneity in productivity-driver relationships. A combination of macro- and microscale models provided the best explanation of site index. Results further showed that site index is mainly driven by species composition (complementarity effects of aspen and jack pine compositions) and stand diameter structural diversity effects. It is concluded that successional changes, more than direct climatic effects, drive productivity.     

Detecting dominant landscape objects through multiple scales: An integration of object-specific methods and watershed segmentation

Complex systems, such as landscapes, are composed of different critical levels of organization where interactions are stronger within levels than among levels, and where each level operates at relatively distinct time and spatial scales. To detect significant features occurring at specific levels of organization in a landscape, two steps are required. First, a multiscale dataset must be generated from which these features can emerge. Second, a procedure must be developed to delineate individual image-objects and identify them as they change through scale. In this paper, we introduce a framework for the automatic definition of multiscale landscape features using object-specific techniques and marker-controlled watershed segmentation. By applying this framework to a high-resolution satellite scene, image-objects of varying size and shape can be delineated and studied individually at their characteristic scale of expression. This framework involves three main steps: 1) multiscale dataset generation using an object-specific analysis and upscaling technique, 2) marker-controlled watershed transformation to automatically delineate individual image-objects as they evolve through scale, and 3) landscape feature identification to assess the significance of these image-objects in terms of meaningful landscape features. This study was conducted on an agro-forested region in southwest Quebec, Canada, using IKONOS satellite data. Results show that image-objects tend to persist within one or two scale domains, and then suddenly disappear at the next, while new image-objects emerge at coarser scale domains. We suggest that these patterns are associated to sudden shifts in the entire image structure at certain scale domains, which may correspond to critical landscape thresholds.This revised version was published online in May 2005 with corrections to the Cover Date.     

Interaction between hHBrk1 and WAVE1 protein

AIM: To identify the binding sites between hHBrk1 and WAVE1 protein.METHODS: PCR was applied to clone the WAVE1 gene and its truncated domains from human fetal brain library. The gene fragments were sub-cloned into eukaryotic expression vector. Co-immunoprecipitation assay was used to identify the binding sites between hHBrk1 and WAVE1.RESULTS: The sequences of WAVE1 and the truncated domains were successfully amplified and sub-cloned into pCMV-HA vector. An HA tag was added to the fused protein at N-terminal. The interaction between hHBrk1 and the Scar homology domain(SHD) of WAVE1 was identified by co-immunoprecipitation. We also observed that WAVE1 bound to the heptad repeat(HR) domain of hHBrk1, and the site-directed mutations at HR domain abolished the interaction between hHBrk1 and WAVE1.CONCLUSION: hHBrk1 may play an important role in migration of tumor cells through binding to SHD of WAVE1 protein.     

Detection,cloning and expression of bone morphogenetic protein-1 from human osteosarcoma cell lines

AIM:To manufacture recombinant protein of the highly conserved domain in human bone morphogenetic protein-1(BMP-1) using gene engineering methods as antigen for making wide spectrum antibody to BMP-1.METHODS:We analyzed the gene sequences and protein structures of BMP-1 and its related proteins, and chose a highly conserved fragment as target gene. Total RNA was prepared from human osteosarcoma cell line Saos-2, then the target gene was amplified with RT-PCR. The PCR product was cloned into prokaryotic expression vector pMAL c2 to get recombinant vector BMP-1(322-588aa)-pMAL c2. After transforming the recombinant plasmid into DH5-alpha and screening, several prositive clones were got for sequencing. Finally the transformed cells was induced with IPTG to get fusion protein.RESULTS:The BMP-1 gene fragment was successfully cloned into vector pMAL c2, and was able to express efficiently with IPTG inducement. The amount of expressed fusion protein is about 66%-72% in total volume of bacterial proteins.CONCLUSIONS:The recombinant protein contains several key domains(2 CUB domains and 1 EGF domain), which are shared by BMP-1 and its related proteins. Specific wide spectrum antibody to human BMP-1 and its related proteins may be generated with this recombinant protein antigen.     

光皮桦BBX类基因BlCOL13的克隆与表达分析

利用RACE、RT-PCR以及基因组步移技术克隆得到光皮桦BlCOL13全长cDNA（GenBank登录号：KP663425）、全长DNA及其启动子序列。该基因DNA全长2 808 bp,具有4个外显子,3个内含子;cDNA全长1 370 bp,包含1 140 bp的开放阅读框,编码含379个氨基酸的蛋白。BlCOL13蛋白N、C端分别有两个B-box和一个CCT结构域,属于典型的BBX蛋白。以拟南芥中BBX蛋白的分类为参照,通过聚类分析表明BlCOL13属于Ⅱ型BBX蛋白;克隆得到的BlCOL13启动子序列长685 bp,含有多个与成花、光响应、激素信号转导以及逆境胁迫相关的顺式作用元件。亚细胞定位试验结果表明该基因主要在细胞核中表达。不同组织中该基因都有一定程度的表达,但在雄花中表达量最高。低温、干旱、ABA和盐胁迫等非生物逆境都在一定程度上诱导BlCOL13的表达,盐胁迫的影响最为明显,表明该基因在光皮桦逆境响应中可能发挥比较重要的作用。     

莲藕可溶性淀粉合成酶基因LrSSS的克隆与表达特性分析

以莲藕品种‘美人红’叶片为试材,利用RACE结合RT-PCR技术克隆得到全长4 080 bp的莲藕可溶性淀粉合成酶基因（LrSSS）cDNA序列（GenBank登录号：KP201636）,其中开放阅读框3 696 bp,编码1 231个氨基酸;该序列与甜瓜、葡萄SSS基因编码氨基酸序列同源性较高,分别达79%、69%。LrSSS 所编码的氨基酸序列包含3个典型的碳水化合物结合结构域（CBM_25）和1个淀粉合成酶催化域（Glyco_transf_5）。LrSSS 表达分析表明,莲藕根状茎膨大具3节段时,在终止叶叶片中的表达量最高,其次为后把叶叶片,终止叶叶柄表达量最少;在根状茎膨大至4节段时,LrSSS在第1、2节段根状茎中表达量较高,在第3、4节段根状茎中表达量较低,表明在第1、2节段根状茎形态基本建成后LrSSS在调控产物转化为淀粉过程中可能起到重要作用。     

菊花胚发育相关基因Cm2S的克隆与表达分析

通过对菊花（Chrysanthemum morifolium）品种‘雨花落英’与四倍体菊花脑（C. nankingense）杂交后胚的转录组和蛋白组数据分析，筛选到1个菊花胚不同发育时期差异表达的基因Cm2S。采用高保真PCR技术，克隆得到Cm2S的开放阅读框（ORF）。序列分析表明，Cm2S开放阅读框为852 bp，编码283个氨基酸。利用SMART在线网站预测Cm2S蛋白的功能结构域，结果显示，Cm2S蛋白属于AAI_LTSS家族，具有两个保守的AAI（Alpha-Amylase Inhibitors）结构域。对其进行同源性比对及系统进化树分析发现，Cm2S与青蒿（Artemisia annua）种子贮藏蛋白AaSSP6亲缘关系最近。亚细胞定位结果显示，Cm2S蛋白在细胞质和细胞核中均有分布。荧光实时定量PCR分析表明，Cm2S在菊花的胚中特异性表达，且在授粉后18 d正常胚中的表达量显著高于授粉后12 d的胚，是其315.11倍，同时也显著高于授粉后18 d败育的胚，是其1.98倍。     

苹果TIFY家族基因鉴定及其在虫害胁迫下的表达分析

利用生物信息学方法在苹果全基因组中鉴定了TIFY转录因子家族基因,对基因结构、蛋白保守基序和进化关系进行了分析,同时基于新疆野苹果(Malus sieversii)转录组数据分析,明确了苹果TIFY家族基因在虫害胁迫条件下的差异表达情况。共鉴定到了16个苹果TIFY基因,其蛋白质大小介于209~449 aa之间。16个TIFY基因分布于苹果8条染色体中,所有的MdTIFY蛋白都具有保守的TIFY结构域。其中8个苹果MdTIFY与新疆野苹果转录组测序拼接转录本对应,这些基因均含有保守的TIFY功能域,多物种进化分析表明该类蛋白可聚类成7个组。7个TIFY基因的表达量在不同处理间表现出显著差异,虫害侵染后,抗虫株系中MsTIFY10B-a表达量升高34倍,MsTIFY9-c上升5.2倍。同时抗虫株系在自然条件下茉莉酸—异亮氨酸含量显著高于敏感株系。新疆野苹果中重要抗虫响应基因MsTIFY10B-a和MsTIFY9-c位于预测关联网络节点中心位置,使它们在调节植物对生物胁迫的响应中发挥重要作用。