植物β-半乳糖苷酶

β-半乳糖苷酶是一个与细胞壁降解相关的酶,广泛分布于植物组织中,参与一系列的生理生化过程,如植物的花粉发育、果实成熟及生长过程中多糖的裂解。目前,已从多种植物中分离到β-半乳糖苷酶基因。β-半乳糖苷酶基因属于多基因家族,随着研究的深入,其不同水平的转录本在不同植物的不同组织中被发现。但目前β-半乳糖苷酶在植物发育中确切的作用机制尚不明确。现介绍目前这一领域内细胞与分子生物学方面的研究进展,并结合所在课题组的研究结果进行相关探讨,为进一步研究β-半乳糖苷酶在植物中的作用机制提供新的线索。     

赤豆子叶β-半乳糖苷酶的纯化及其性质的研究

β-半乳糖苷酶 ( EC3.2 .1 .2 )广泛存在于动植物的组织中 ,如在杏仁、桃子、大豆、咖啡豆等植物 ,蜗牛 ,哺乳动物的肠道中都有 β-半乳糖苷酶 .同样 ,微生物也能产生β-半乳糖苷酶 ,俗称乳糖酶 .乳糖操纵子学说的提出就是建立在对微生物β-半乳糖苷酶研究基础之上的 .在过去的研究中 ,关于微生物、动物来源的乳糖酶报道较多[1] ,而对于植物来源的β-半乳糖苷酶研究报道却相对较少[2 ] .它可能降解多糖中 β-构型半乳糖苷键 ,为种子生长发育提供必要的能量来源 .但目前对β-半乳糖苷酶在植物中确切的生理生化功能尚不清楚 .为了进一步阐明…     

香蕉果实软化相关β-半乳糖苷酶基因的克隆及其表达分析

β-半乳糖苷酶（β-galactosidase）通过分解细胞壁半纤维素切除半乳糖键而参与果实软化。为了阐明香蕉（Musasp．）果实成熟过程中的软化与细胞壁代谢酶β-半乳糖苷酶基因表达之间的关系,采用RT-PCR方法,从成熟香蕉果实果肉中分离了编码β-半乳糖苷酶基因的部分cDNA（MA-Gal）,序列分析表明,MA-Gal包含927bp,编码309个氨基酸,包含5个β-半乳糖苷酶结构域（典型真核生物中β-半乳糖苷酶包含7个结构域）,推导的MA-Gal蛋白质中有β-半乳糖苷酶蛋白的催化活性部位GGPIILSQIENEY（F）;系统进化树分析结果表明MA-Gal属于第一类β-半乳糖苷酶基因（该类主要在果实中表达）;β-半乳糖苷酶活性和硬度的变化表明其与香蕉果实硬度变化密切相关;Northern分析显示,跃变前期的果肉中,MA-Gal基因的表达量很低,后随着果实的软化表达量不断增加,并在呼吸跃变后达到最高。所有结果表明,MA-Gal参与香蕉果实成熟过程中的软化。     

表达β-半乳糖苷酶的gal B16肿瘤模型的建立及其在肿瘤DNA疫苗研究中的应用

本文工作的目的是建立以β-半乳糖苷酶为标志性抗原的小鼠黑色素瘤模型,并进行肿瘤免疫的研究。我们首先在pcDNA3质粒中引入一个β-半乳糖苷酶编码基因从而建立转染质粒p3gal。p3gal转染小鼠黑色素瘤细胞B16后,再通过G418筛选及X-Gal细胞染色得到表达β-半乳糖苷酶的gal B16细胞株。接着用该细胞株成功地在C57小鼠上建立了表达β-半乳糖苷酶的gal B16肿瘤模型。并在此模型上观察了β-半乳糖苷酶编码基因作为DNA疫苗抑制gal B16肿瘤生长的作用。     

表达β-半乳糖苷酶的gal B16肿瘤模型的建立及其在肿瘤DNA疫苗研究中的应用

本文工作的目的是建立以β-半乳糖苷酶为标志性抗原的小鼠黑色素瘤模型,并进行肿瘤免疫的研究.我们首先在pcDNA3质粒中引入一个β-半乳糖苷酶编码基因从而建立转染质粒p3gal.p3gal转染小鼠黑色素瘤细胞B16后,再通过G418筛选及X-Gal细胞染色得到表达β-半乳糖苷酶的galB16细胞株.接着用该细胞株成功地在C57小鼠上建立了表达β-半乳糖苷酶的galB16肿瘤模型.并在此模型上观察了β-半乳糖苷酶编码基因作为DNA疫苗抑制galB16肿瘤生长的作用.     

β-半乳糖苷酶的筛选、克隆表达、酶学性质及其酶法合成低聚半乳糖

采用人工底物邻硝基苯酚-β-D-半乳糖苷(o NPG)为筛选标记,从耐有机溶剂微生物菌库中,筛选出具有较高水解活性的β-半乳糖苷酶产生菌,再以乳糖为底物考察菌株低聚半乳糖的合成性能,筛选得到1株产β-半乳糖苷酶的Erwinia billingiae WX1。根据Gen Bank中相同属种的基因组序列推测β-半乳糖苷酶基因,克隆得到β-半乳糖苷酶基因gal,并在大肠杆菌中实现了来源于Erwinia billingiae菌β-半乳糖苷酶的克隆表达。该基因的开放阅读框(ORF)为1 428 bp,编码475个氨基酸,理论相对分子质量为5.2×104。镍柱法分离纯化得到电泳纯的β-半乳糖苷酶GAL,其酶学性质研究表明最适催化温度55℃,最适p H 7.0;Mg~(2+)、Mn~(2+)对该酶起较强促进作用,EDTA对该酶抑制作用较强。利用β-半乳糖苷酶GAL的转糖基作用,以乳糖为底物合成低聚半乳糖,初步优化的反应条件:底物乳糖质量浓度400 g/L,每克乳糖添加酶量1.0 U,在40℃反应16 h后,低聚半乳糖合成率达到34%(质量分数),显示了较好的开发前景。     

棉花半乳糖苷酶基因启动子的分离及其在转基因烟草中的表达

β-半乳糖苷酶(β-galactosidase, EC 3.2.1.23)由植物中广泛分布的一类糖基水解酶组成, 被认为与细胞壁多糖的代谢相关. 棉花(Gossypium hirsutum) β-半乳糖苷酶基因已被成功分离, 被命名为GhGal1. RNA杂交实验显示该基因在棉花纤维发育的伸长期优势表达. 为了分析GhGal1基因的时空表达调控, 本研究构建了GhGal1启动子区域(1770 bp)与β-葡糖醛酸糖苷酶(glucuronidase, GUS)基因融合的双元载体, 通过农杆菌转化烟草植株. 对转基因植株分析的结果表明: 此转基因果实中的GUS活性比阴性和阳性对照的活性高. GUS组织定位分析表明: β-半乳糖苷酶基因能在根组织的分生区、子叶、维管束组织、果实和表皮毛中表达. 此外, 调控区域的序列分析揭示该序列含有一些果实/种子特异表达以及与表皮毛表达相关的保守元件. 这些结果显示了GhGal1启动子在转基因烟草植株中的时空表达特征, 并提供了GhGal1基因参与棉花纤维发育的一些重要线索.     

微生物源α-半乳糖苷酶的研究进展

介绍了微生物源α-半乳糖苷酶的生理生化特性、合成调控机制的研究进展情况及其在食品、饲料、医药工业等领域的一些应用。Α-半乳糖苷酶均是糖蛋白,不同来源的α-半乳糖苷酶的作用基质特异性差别较大,作用基质特异性差别是由蛋白质部分N-末端氨基酸序列决定的。不同微生物来源的α-半乳糖苷酶其最佳作用条件、pH稳定性及耐热性差异较大。微生物α-半乳糖苷酶是一种诱导酶,其合成受多个基因的调控,高浓度的葡萄糖能抑制其合成。     

假单胞菌M18转录调节因子σs基因与无启动子lacZ基因融合突变株的构建和鉴定

通过菌落原位杂交和Southern杂交,从假单胞菌M18基因组文库中克隆了rpoS基因及相邻序列。为了深入研究影响rpoS基因表达的调控因素,运用同源重组技术,将无启动子β-半乳糖苷酶基因(-′lacZ)插入并融合于rpoS基因中,构建了假单胞菌M18rpoS基因突变株M18SZ。Miller法测定显示,突变株M18SZ的β-半乳糖苷酶可高达480U,而野生株检测不到β-半乳糖苷酶活性。表明,突变株中的rpoS基因与无启动子β-半乳糖苷酶基因已融合并且表达。在KMB培养基中生长量测定(OD600)的结果表明,突变株与野生株生长存在显著差异。     

微生物α-半乳糖苷酶的研究进展

α-半乳糖苷酶在多种生物内广泛存在,微生物是目前α-半乳糖苷酶的主要来源。微生物α-半乳糖苷酶可按照底物特异性或序列同源性分类,在古菌、细菌和真菌中均存在,其性质与来源和家族有关,催化机理大多为构型保留机制,目前主要应用于食品与饲料工业,还可用于生物质降解和医药领域。展望了微生物α-半乳糖苷酶的研究趋势。本文对相关研究者具有一定的参考意义。     

Biochemical characterization of a strictly specific beta-galactosidase from the digestive juice of the palm weevil Rhynchophorus palmarum larvae

A beta‐galactosidase from the digestive juice of the palm weevil Rhynchophorus palmarum L. larvae was purified by chromatography on ion exchange, gel filtration, and hydrophobic interaction columns. The preparation was shown to be homogeneous on polyacrylamide gel. Beta‐galactosidase was a monomeric protein with a molecular weight of 62 kDa based on its mobility in sodium dodecyl sulfate–polyacrylamide gel electrophoresis and 60 kDa based on gel filtration. Maximal enzyme activity occurred at 55°C and pH 5.0. The purified beta‐galactosidase was stable at 37°C and its pH stability was in the range of 4.6–6.0. Beta‐galactosidase was highly specific for the beta‐d ‐galactosyl residue and beta‐(1‐4) linkage. The catalytic efficiency (V_max/K_m) values for p‐nitrophenyl‐beta‐d ‐galactopyranoside, beta‐d ‐galactosyl(1‐4)‐d ‐glucose (lactose), beta‐d ‐galactosyl(1‐4)‐d ‐galactose and beta‐d ‐galactosyl(1‐4)‐beta‐d ‐galactosyl(1‐4)‐d ‐glucose were, respectively, 72.95, 10.97, 20.74 and 12.73. 5,5‐Dithio‐bis(2‐nitrobenzoate) and sodium dodecyl sulfate inhibited completely the beta‐galactosidase activity. The enzyme was capable of catalyzing transgalactosylation reactions. The yield of galactosylation of 2‐phenylethanol (43%), catalyzed by the beta‐galactosidase in the presence of lactose as galactosyl donor, is higher than those reported previously with conventional sources of beta‐galactosidases. In addition, the optimum pH is different for the hydrolysis (pH 5.0) and transgalactosylation reactions (pH 6.0).     

Novel genes underlying beta cell survival in metabolic stress

Relative insulin deficiency, in response to increased metabolic demand (obesity, genetic insulin resistance, pregnancy and aging) lead to Type2 diabetes. Susceptibility of the type 2 diabetes has a genetic basis, as a subset of people with risk factors (obesity, Insulin Resistance, pregnancy), develop Type2 Diabetes. We aimed to identify ‘cluster’ of overexpressed genes, underlying increased beta cell survival in diabetes resistant C57BL/6J ob/ob mice (compared to diabetes susceptible BTBR ob/ob mice). We used ‘consensus’ overexpression status to identify ‘cluster’ of 11 genes consisting of Aldh18a1, Rfc4, Dynlt3, Prom1, H13, Psen1, Ssr4, Dad1, Anpep, Fam111a and Plk1. Information (biological processes, molecular functions, cellular components, protein-protein interactions/associations, gene deletion/knockout/inhibition studies) of all the genes in ‘cluster’ were collected by text mining using different literature search tools, gene information databases and protein-protein interaction databases. Beta cell specific function of these genes were also inferred using meta analysis tool of Beta Cell Biology Consortium, by studying the expression pattern of these genes in microarray studies related to beta-cell stimulation/injury, pancreas development and growth and cell differentiation. In the ‘clusters’, 6 genes (Dad1, Psen1, Ssr4, Rfc4, H13, Plk1) have a role in cell survival. Only Psen1 was previously identified to have role in successful beta cell compensation. We advocate these genes to be potentially involved in successful beta cell compensation and prevent T2D in humans, by conferring protection against diabetogenic insults.     

Generation of stable cell line by using chitosan as gene delivery system

Establishing stable cell lines are useful tools to study the function of various genes and silence or induce the expression of a gene of interest. Nonviral gene transfer is generally preferred to generate stable cell lines in the manufacturing of recombinant proteins. In this study, we aimed to establish stable recombinant HEK-293 cell lines by transfection of chitosan complexes preparing with pDNA which contain LacZ and GFP genes. Chitosan which is a cationic polymer was used as gene delivery system. Stable HEK-293 cell lines were established by transfection of cells with complexes which were prepared with chitosan and pVitro-2 plasmid vector that contains neomycin drug resistance gene, beta gal and GFP genes. The transfection efficiency was shown with GFP expression in the cells using fluorescence microscopy. Beta gal protein expression in stable cells was examined by beta-galactosidase assay as enzymatically and X-gal staining method as histochemically. Full complexation was shown in the above of 1/1 ratio in the chitosan/pDNA complexes. The highest beta-galactosidase activity was obtained with transfection of chitosan complexes. Beta gal gene expression was 15.17 ng/ml in the stable cells generated by chitosan complexes. In addition, intensive blue color was observed depending on beta gal protein expression in the stable cell line with X-gal staining. We established a stable HEK-293 cell line that can be used for recombinant protein production or gene expression studies by transfecting the gene of interest.     

Islet dynamics: a glimpse at beta cell proliferation

Pancreatic islets consist of 60-80% beta cells, which secrete insulin, a hormone of profound importance in the regulation of carbohydrate, fat and protein metabolism. Beta cell death and/or dysfunction result in an insufficient amount of insulin that leads to high glucose levels in the blood, a metabolic disorder known as Diabetes mellitus. Many studies aiming to establish new therapeutic applications for this disorder are targeted at understanding and manipulating the mechanisms of beta cell proliferation and function. The present comprehensive review summarizes the advances in the field of beta cell renewal and focuses on three fundamental issues: (i) identification of the cellular origins of new beta cells in the adult, (ii) regulation of beta cell proliferation, and (iii) downstream signaling events controlling the cell cycle machinery. Although the source of new adult beta cells is still being debated, recent findings in mice show an important contribution of beta cell proliferation to adult beta cell mass. In conjunction with describing characterized beta cell mitogens and components of the beta cell cycle machinery, we discuss how manipulating the proliferative potential of beta cells could provide novel methods for expanding beta cell mass. Such an expansion could be achieved either through in vitro systems, where functional beta cells could be generated, propagated and further used for transplantation, or in vivo, through directed beta cell renewal from sources in the organism. Once established, these methods would have profound benefits for diabetic patients.     

A novel bioinformatics approach identifies candidate genes for the synthesis and feruloylation of arabinoxylan

Arabinoxylans (AXs) are major components of graminaceous plant cell walls, including those in the grain and straw of economically important cereals. Despite some recent advances in identifying the genes encoding biosynthetic enzymes for a number of other plant cell wall polysaccharides, the genes encoding enzymes of the final stages of AX synthesis have not been identified. We have therefore adopted a novel bioinformatics approach based on estimation of differential expression of orthologous genes between taxonomic divisions of species. Over 3 million public domain cereal and dicot expressed sequence tags were mapped onto the complete sets of rice (Oryza sativa) and Arabidopsis (Arabidopsis thaliana) genes, respectively. It was assumed that genes in cereals involved in AX biosynthesis would be expressed at high levels and that their orthologs in dicotyledonous plants would be expressed at much lower levels. Considering all rice genes encoding putative glycosyl transferases (GTs) predicted to be integral membrane proteins, genes in the GT43, GT47, and GT61 families emerged as much the strongest candidates. When the search was widened to all other rice or Arabidopsis genes predicted to encode integral membrane proteins, cereal genes in Pfam family PF02458 emerged as candidates for the feruloylation of AX. Our analysis, known activities, and recent findings elsewhere are most consistent with genes in the GT43 families encoding beta-1,4-xylan synthases, genes in the GT47 family encoding xylan alpha-1,2- or alpha-1,3-arabinosyl transferases, and genes in the GT61 family encoding feruloyl-AX beta-1,2-xylosyl transferases.     

The interferon system and interferon regulatory factor transcription factors -- studies from gene knockout mice

Interferon regulatory factors (IRFs) were initially identified as regulators of IFN-alpha/beta genes and to date nine members have been determined in human and mouse. They share a conserved DNA-binding domain in the N-terminal portion that recognizes similar DNA sequences. Despite their similar DNA binding specificity, the IRFs show diverse functions in response to extra cellular stimuli. Although the study of IRFs was started with respect to regulation of the IFN-alpha/beta gene expression, recent studies have revealed other aspects of IRF functions. In this review, we summarize our current knowledge of the functions of IRF family members revealed by our gene targeting study in mice, focusing on the regulation of the IFN system.     

Anion channels in higher plants: functional characterization, molecular structure and physiological role

Anion channels are well documented in various tissues, cell types and membranes of algae and higher plants, and current evidence supports their central role in cell signaling, osmoregulation, plant nutrition and metabolism. It is the aim of this review to illustrate through a few selected examples the variety of anion channels operating in plant cells and some of their regulation properties and unique physiological functions. In contrast, information on the molecular structure of plant anion channels has only recently started to emerge. Only a few genes coding for putative plant anion channels from the large chloride channel (CLC) family have been isolated, and current molecular data on these plant CLCs are presented and discussed. A major challenge remains to identify the genes encoding the various anion channels described so far in plant cells. Future prospects along this line are briefly outlined, as well as recent advances based on the use of knockout mutants in the model plant Arabidopsis thaliana to explore the physiological functions of anion channels in planta.