在杂交作物分子育种中利用普通核雄性不育的几个可能途径

由一对隐性基因控制的普通核雄性不育性遗传方式能够满足对植物最佳雄性不育系选育的要求,是水稻等作物杂种优势利用的极好遗传工具。如果能解决其不育系繁殖问题,将优于现有的其他杂种优势利用方式。克隆出普通核雄性不育性的可育基因,通过叶绿体转化,将核雄性不育性可育基因向普通核雄性不育株细胞质转移,创造普通核雄性不育株的保持系;通过种子成熟后表达的启动子;和以位点特异性重组技术为基础的基因开关以及化学诱导启动子的利用,都可能繁殖出100%不育株率的普通核雄性不育系,创造普通核雄性不育性利用的新途径,对植物杂种优势利用产业有十分重要的意义。     

在杂交作物分子育种中利用普通核雄性不育的几个可能途径

由一对隐性基因控制的普通核雄性不育性遗传方式能够满足对植物最佳雄性不育系选育的要求,是水稻等作物杂种优势利用的极好遗传工具。如果能解决其不育系繁殖问题,将优于现有的其他杂种优势利用方式。克隆出普通核雄性不育性的可育基因,通过叶绿体转化,将核雄性不育性可育基因向普通核雄性不育株细胞质转移,创造普通核雄性不育株的保持系;通过种子成熟后表达的启动子;和以位点特异性重组技术为基础的基因开关以及化学诱导启动子的利用,都可能繁殖出100％不育株率的普通核雄性不育系,创造普通核雄性不育性利用的新途径,对植物杂种优势利用产业有十分重要的意义。     

基因捕捉及其在植物基因分离和功能基因组学上的应用

基因捕捉是一种报告基因的随机整合技术。基因捕捉系统已成为分离基因、鉴定基因功能的重要手段。基因捕捉(gene traps)包括增强子捕捉(enhancer trap)、启动子捕捉(promoter trap)和基因捕捉(gene trap),通称为基因捕捉(gcne traps)。在增强子捕捉中,报告基因与一个基本启动子融合,这个启动子不能使报告基因表达,但可被临近的增强子激活。在启动子捕捉和基因捕捉中,报告基因的启动子被去除,融合基因只有以正确的方向插入到转录单元内才能表达。对基因捕捉系统的结构特征、构建方法、应用范围、研究现状和应用前景等作了系统论述,并对有关问题进行了讨论。     

Differentiation genes: are they primary targets for human carcinogenesis?

In spite of extensive research in molecular carcinogenesis, genes that can be considered primary targets in human carcinogenesis remain to be identified. Mutated oncogenes or cellular growth regulatory genes, when incorporated into normal human epithelial cells, failed to immortalize or transform these cells. Therefore, they may be secondary events in human carcinogenesis. Based on some experimental studies we have proposed that downregulation of a differentiation gene may be the primary event in human carcinogenesis. Such a gene could be referred to as a tumor-initiating gene. Downregulation of a differentiation gene can be accomplished by a mutation in the differentiation gene, by activation of differentiation suppressor genes, and by inactivation of tumor suppressor genes. Downregulation of a differentiation gene can lead to immortalization of normal cells. Mutations in cellular proto-oncogenes, growth regulatory genes, and tumor suppressor genes in immortalized cells can lead to transformation. Such genes could be called tumor-promoting genes. This hypothesis can be documented by experiments published on differentiation of neuroblastoma (NB) cells in culture. The fact that terminal differentiation can be induced in NB cells by adenosine 3',5'-cyclic monophosphate (cAMP) suggests that the differentiation gene in these cells is not mutated, and thus can be activated by an appropriate agent. The fact that cAMP-resistant cells exist in NB cell populations suggests that a differentiation gene is mutated in these cancer cells, or that differentiation regulatory genes have become unresponsive to cAMP. In addition to cAMP, several other differentiating agents have been identified. Our proposed hypothesis of carcinogenesis can also be applied to other human tumors such as melanoma, pheochromocytoma, medulloblastoma, glioma, sarcoma, and colon cancer.     

高密度基因芯片探针布局方法

为了提高基因芯片制备质量和检测的准确性,提出两种基因芯片布局方法,一是分子印章凸点优化布局方法,另一种是基于探针杂交解链温度的梯度场布局方法。利用上述两种方法对所设计的高密度基因芯片进行控针布局实验,结果表明,第一种方法能够使制备基因芯片的分子印章上凸点均匀分布,解决误压印问题,从而提高基因芯片的制备质量;而第二种方法能够使基因芯片上的探针按照杂交解链温度有序地组织起来,从而提高基因芯片对碱基错配的辨别力。     

Diploidy, evolution and sex

A new gene for a new purpose may be created by mutation of a pre-existing gene. But if that original gene is still required for its original purpose, and is to be retained side by side with the new, a spare copy is needed initially as raw material for the innovation. Thus in haploids the original gene must be duplicated before it is modified. But in diploids a spare copy of every gene is always available, and a mutant allele serving a new purpose can be easily established and maintained by heterosis in parallel with the old allele. Subsequent gene duplication will lead, via crossing-over, to insertion of the new gene in tandem with the old, as a permanent addition to the genome. Calculations show that diploids can thus enlarge their genomes with new genes for new purposes much more readily than haploids; in particular, they can more easily evolve the complex gene control systems characteristic of differentiated multicellular organisms. Sexual reproduction preserves diploidy, and so can be seen as the basis of these richer possibilities for evolutionary innovation.     

Untangling multi-gene families in plants by integrating proteomics into functional genomics

The classification and study of gene families is emerging as a constructive tool for fast tracking the elucidation of gene function. A multitude of technologies can be employed to undertake this task including comparative genomics, gene expression studies, sub-cellular localisation studies and proteomic analysis. Here we focus on the growing role of proteomics in untangling gene families in model plant species. Proteomics can specifically identify the products of closely related genes, can determine their abundance, and coupled to affinity chromatography and sub-cellular fractionation studies, it can even provide location within cells and functional assessment of specific proteins. Furthermore global gene expression analysis can then be used to place a specific family member in the context of a cohort of co-expressed genes. In model plants with established reverse genetic resources, such as catalogued T-DNA insertion lines, this gene specific information can also be readily used for a wider assessment of specific protein function or its capacity for compensation through assessing whole plant phenotypes. In combination, these resources can explore partitioning of function between members and assess the level of redundancy within gene families.     

Development of DNA-mediated transformation systems for plants

The genetic engineering of plants by DNA-mediated gene transfer requires that efficient transformation systems be developed. Considerable progress has been made in manipulating the Ti plasmid of Agrobacterium tumefaciens as a vehicle for delivery of foreign genes into protoplasts of dicotyle-donous plants. Part of the Ti plasmid, the T-DNA, can be incorporated into the genome of the host cell; the T-DNA can carry a foreign DNA sequence which co-integrates with it; under normal conditions, the tumorigenic-causing portion of the T-DNA can be inactivated so that transformed protoplasts can be regenerated and T-DNA with an inserted foreign gene can be stably maintained during regeneration, meiosis and gamete formation. A foreign gene has yet to be expressed in regenerated plants although a T-DNA gene for opine synthesis can function in regenerates. Developing a more ubiquitous transformation system for monocotyledons is further from fruition. Based on transformation systems for simple eukaryotic organisms, it is reasonable to expect that a DNA vector which is capable of amplifying a novel plant gene and which contains both a drug resistance marker to facilitate the selection of transformed plant protoplasts and a species-specific autonomously replicating sequence to ensure the stable maintenance of the input gene in the recipient cell can be constructed.     

Adaptive noise

In biology, noise implies error and disorder and is therefore something which organisms may seek to minimize and mitigate against. We argue that such noise can be adaptive. Recent studies have shown that gene expression can be noisy, noise can be genetically controlled, genes and gene networks vary in how noisy they are and noise generates phenotypic differences among genetically identical cells. Such phenotypic differences can have fitness benefits, suggesting that evolution can shape noise and that noise may be adaptive. For example, gene networks can generate bistable states resulting in phenotypic diversity and switching among individual cells of a genotype, which may be a bet hedging strategy. Here, we review the sources of noise in gene expression, the extent to which noise in biological systems may be adaptive and suggest that applying evolutionary rigour to the study of noise is necessary to fully understand organismal phenotypes.     

Magnetic resonance-based visualization of gene expression in mammalian cells using a bacterial polyphosphate kinase reporter gene

Gene expression reporter systems, in which a promoter of interest is cloned upstream of a readily assayed reporter gene, have been developed and used extensively to study gene expression in prokaryotes and eukaryotes. Unfortunately, most of these systems cannot be used to assay gene expression in nonsuperficial tissues in living organisms. This study examines a novel reporter gene system based on the gene encoding Escherichia coli polyphosphate kinase (PPK), which can be used to monitor gene expression in mammalian cells. PPK catalyzes the synthesis of inorganic polyphosphate (polyP) from ATP, and because mammalian cells do not contain detectable levels of polyP, PPK activity can be measured in mammalian cells using 31P-magnetic resonance spectroscopy or 31P-magnetic resonance imaging. The ppk reporter gene system described here is noninvasive, does not require an exogenous substrate, and can potentially be used in internal tissues of living organisms.     

抗菌肽Cecropin B-人溶菌酶融合蛋白表达载体的构建

目的是构建抗菌肽B(Cecropin B)和人溶菌酶(hLyso)的融合蛋白表达载体。从pUC118～hLyso上卸下人溶菌酶基因后,通过重叠区扩增法人工合成抗菌肽B基因,并将其融合到人溶菌酶基因的5’端。将抗菌肽B基因和人溶菌酶基因按正确的阅读框架定向克隆至大肠杆菌高效表达载体pET32a,终止子位于人溶菌酶基因的3’端。PCR鉴定及序列分析表明,所转化的BL21(DE3)菌落中含有插入Cecropin B-hLyso基因的重组质粒pET32a-CB-hLyso。     

Gene fusion vectors based on the gene for staphylococcal protein A

Two plasmid vectors, containing the gene coding for staphylococcal protein A and adapted for gene fusion, have been constructed. These vectors will allow fusion of any gene to the protein A gene, thus giving hybrid proteins which can be purified, in a one-step procedure, by IgG affinity chromatography. As an example of the practical use of such vectors, the protein A gene has been fused to the lacZ gene of Escherichia coli. E. coli strains containing such plasmids produce hybrid proteins with both IgG binding and β-galactosidase activities. The hybrid protein(s) can be immobilized on IgG-Sepharose by its protein A moiety with high efficiency without losing its enzymatic activity and they can be eluted from the column by competitive elution with pure protein A. The fused protein(s) also binds to IgG-coated microtiter wells which means that the in vivo product can be used as an enzyme conjugate in ELISA tests.     

Inducible gene expression by retrovirus-mediated transfer of a modified tetracycline-regulated system.

The ability to regulate gene expression via exogenous stimuli will facilitate the study of gene functions in mammalian cells. In the present study, we modified the tetracycline-controlled inducible system by the addition of the ligand-binding domain of the estrogen receptor to the carboxy terminus of the tTA transactivator. A single retroviral vector can transduce both the transactivator gene and the gene of interest controlled by the tTA-inducible promoter into mammalian cells. We show that cell lines expressing the transactivator can readily be established and that expression of the gene of interest depends on the removal of tetracycline and the addition of estrogen. By using this system, cell lines with inducible expression of the G protein of vesicular stomatitis virus, a potentially toxic gene product, were established. The combination of a powerful inducible system and retrovirus-mediated gene transfer can not only be used to study gene function but may also be applied in the future to clinical trials in human gene therapy.     

siRNA介导基因沉默的分子机器、作用模式和应用前景

siRNA(small interfering RNA)介导的基因沉默是细胞内监控寄生的遗传物质、沉默无用的信息模板和调节自然的时空转换的一种分子机制。它与体液免疫和细胞免疫一起形成了动物和人类机体中免疫系统的三大支柱,从不同水平上来对抗体内外有害物质的干扰和侵犯。现已清楚,大约22个核苷酸长的双链RNA能够通过不同途径,以序列特异的方式来高效地沉默含有同源序列的靶RNA分子。这一古老而又迷人的系统现已被公认为是鉴定基因功能、调控基因表达和改变基因表型的简单而有效的方法。可以预计,这一新颖的技术在不远的将来必将形成一条研究功能基因组学、探索信号通路和创造遗传缺陷模型的高速公路,并将开创一条预防和治疗人类疾病的新途径。     

利用基因诱捕识别哺乳动物发育调控基因

ES细胞是一种来源于胚胎的多潜能细胞,它可在体外培养并进行基因操作,而且通过囊胚注射制作嵌合体的途径,能将外源基因掺入小鼠的基因库中,因此利用ES细胞可筛选出发生基因突变的小概率事件并获得其遗传突变体．利用基因诱捕载体与ES细胞,研究与哺乳动物发育调控有关的未知基因,这一新技术将成为阐明胚胎发育过程中基因表达的时空格式的有效手段．     

dsRNA介导植物基因沉默及其应用

植物双链RNA(double stranded RNA,dsRNA)能有效干扰同源基因的表达,近年来已成为功能基因组学研究上的新方法。本文综述了植物dsRNA介导的转基因沉默现象及其特点、分子作用机制、主要介导方法,以及近年来在植物功能基因组学研究上的应用情况。     

A simple assay for DNA transfection by incubation of the cells in culture dishes with substrates for beta-galactosidase

Transfection efficiency of different cell types as well as promoter strength of cloned genes can be easily determined by direct assay of beta-galactosidase activity encoded from recombinant genes containing the E. coli beta-galactosidase gene. A substrate for beta-galactosidase, o-nitrophenyl-beta-D-galactopyranoside (ONPG), can be added to dishes containing the transfected cells, and the intensity of the colored enzyme product released from either the intact cell or cells lysed in the dishes can be determined. The results obtained by this assay are a reliable measure of transfection efficiency as well as promotor strength of the genes introduced into the cells. In addition, cells expressing the transfected gene can be identified and quantitated under a light microscope after incubation with X-gal. Thus, it is more convenient to use the E. coli beta-galactosidase gene than the chloramphenicol acetyltransferase gene as a reporter gene in the evaluation of DNA transfection.     

外源ble基因在杜氏盐藻中的瞬时表达

利用电击法将带有ble基因的pSP124S转入杜氏盐藻细胞内进行瞬时表达．研究了外源基因在盐藻内的存留及表达情况,确定了合适的电击转化条件,发现利用电击法可以使大量的质粒导入盐藻细胞,质粒在细胞中逐渐降解但至少96h内可以检测得到。外源启动子能够使ble基因有效转录,转录至少可以持续72h,ble基因能够在盐藻细胞中正确翻译,可以作为盐藻遗传转化研究的筛选标记。     

An effective data mining technique for reconstructing gene regulatory networks from time series expression data

Recent development in DNA microarray technologies has made the reconstruction of gene regulatory networks (GRNs) feasible. To infer the overall structure of a GRN, there is a need to find out how the expression of each gene can be affected by the others. Many existing approaches to reconstructing GRNs are developed to generate hypotheses about the presence or absence of interactions between genes so that laboratory experiments can be performed afterwards for verification. Since, they are not intended to be used to predict if a gene in an unseen sample has any interactions with other genes, statistical verification of the reliability of the discovered interactions can be difficult. Furthermore, since the temporal ordering of the data is not taken into consideration, the directionality of regulation cannot be established using these existing techniques. To tackle these problems, we propose a data mining technique here. This technique makes use of a probabilistic inference approach to uncover interesting dependency relationships in noisy, high-dimensional time series expression data. It is not only able to determine if a gene is dependent on another but also whether or not it is activated or inhibited. In addition, it can predict how a gene would be affected by other genes even in unseen samples. For performance evaluation, the proposed technique has been tested with real expression data. Experimental results show that it can be very effective. The discovered dependency relationships can reveal gene regulatory relationships that could be used to infer the structures of GRNs.