病毒诱导的基因沉默技术用于植物色素代谢机制的研究进展

色彩是评价园艺植物观赏性状的重要指标,而植物色素是影响植物色彩表型的关键因子。植物色素及其代谢产物在植物观赏器官颜色形成、植株生长发育调节及对逆境胁迫的响应等方面发挥着重要的作用,是植物研究领域长期关注的热点问题。病毒诱导基因沉默(virus-induced gene silencing,VIGS)是利用植物同源依赖性防御机制,特异性降低宿主内源性基因表达的一种重要基因组学工具,能够通过快速诱导植物基因沉默表型的产生,表征基因的功能,为缺乏遗传转化体系的植物的基因功能鉴定提供高效可行的替代方案。本文综述了VIGS技术在植物色素的生物合成、降解和调控机制上的应用现状,并探讨了VIGS技术在探究色素调控机制上的潜力和未来前景,以期进一步完善对不同植物色素的代谢过程和调控机制的理解,为改良植物色彩性状提供参考依据。     

ABC transporters and RNAi in Caenorhabditis elegans

RNAi is an evolutionarily conserved gene-silencing phenomenon that can be triggered by exogenous delivery of double stranded RNA to organisms. In Caenorhabditis elegans, the response to dsRNA is remarkably robust, and systemic RNAi responses are often observed. We have taken a genetic approach using this organism to better understand the mechanisms that facilitate RNAi. By analyzing strains of RNAi-defective mutants, we have uncovered an unexpected role for ABC transporters in RNAi and related silencing mechanisms. Ten of the sixty ABC transporter genes encoded in the C. elegans genome are required for robust RNAi. We will present data that highlights common features of these genes relative to their roles in RNAi, including genetic interactions with other components of the RNAi machinery. We will also describe unique roles for some transporter genes in endogenous RNAi-related processes.     

病毒诱导烟草的基因沉默

病毒诱导基因沉默是利用RNA介导病毒防卫机制的一项技术。构建含有目的基因片段的人工改造病毒载体,通过农杆菌侵染导致植物内源目的基因沉默。为建立病毒诱导基因沉默体系,选用烟草脆裂病毒（TRV）和烟草为实验材料。构建了八氢番茄红素去饱和酶基因（PDS）的基因沉默病毒载体,病毒载体侵染结果显示目的基因PDS沉默导致烟草幼苗出现光漂白现象。采用RT-PCR的方法检测目的基因PDS的沉默效果,结果显示PDS基因mRNA被显著降解。该体系的建市有利于将来对植物基因进行高通量功能分析。     

Genome Evolution Due to Allopolyploidization in Wheat

The wheat group has evolved through allopolyploidization, namely, through hybridization among species from the plant genera Aegilops and Triticum followed by genome doubling. This speciation process has been associated with ecogeographical expansion and with domestication. In the past few decades, we have searched for explanations for this impressive success. Our studies attempted to probe the bases for the wide genetic variation characterizing these species, which accounts for their great adaptability and colonizing ability. Central to our work was the investigation of how allopolyploidization alters genome structure and expression. We found in wheat that allopolyploidy accelerated genome evolution in two ways: (1) it triggered rapid genome alterations through the instantaneous generation of a variety of cardinal genetic and epigenetic changes (which we termed “revolutionary” changes), and (2) it facilitated sporadic genomic changes throughout the species’ evolution (i.e., evolutionary changes), which are not attainable at the diploid level. Our major findings in natural and synthetic allopolyploid wheat indicate that these alterations have led to the cytological and genetic diploidization of the allopolyploids. These genetic and epigenetic changes reflect the dynamic structural and functional plasticity of the allopolyploid wheat genome. The significance of this plasticity for the successful establishment of wheat allopolyploids, in nature and under domestication, is discussed.     

Influences of electrolytes and glucose on formulation of carbonate apatite nanocrystals for efficient gene delivery to mammalian cells

Genetic manipulation of human cells through delivery of a functional gene or a gene-silencing element is an attractive approach to treat critical diseases very precisely and effectively. Extensive research on the genetic basis of human diseases with complete sequencing of human genome has revealed many vital genes as possible targets in gene therapy programs. On the other hand, to facilitate cell- or tissue-directed delivery of genes and gene-silencing nucleic acid sequences, both genetic and chemical engineering approaches have led to the generation of various viral and nonviral carriers. However, considering the issues of both safety and efficacy, none of the existing vectors is an ideal candidate for clinical use. We recently established pH-sensitive inorganic nanocrystals of carbonate apatite with capability of efficient intracellular delivery and release of associated DNA molecules for subsequent protein expression. Here we show a new synthetic approach for carbonate apatite crystals with stronger affinity toward DNA, leading to significant increment in both transgene delivery and expression. Moreover, CaCl₂ and NaCl, existing as the major electrolytes in the bicarbonate-buffered solution, dose-dependently govern particle size and eventually internalization and expression of particle-associated DNA.     

Genetic and epigenetic interactions in allopolyploid plants

Allopolyploid plants are hybrids that contain two copies of the genome from each parent. Whereas wild and cultivated allopolyploids are well adapted, man-made allopolyploids are typically unstable, displaying homeotic transformation and lethality as well as chromosomal rearrangements and changes in the number and distribution of repeated DNA sequences within heterochromatin. Large increases in the length of some chromosomes has been documented in allopolyploid hybrids and could be caused by the activation of dormant retrotransposons, as shown to be the case in marsupial hybrids. Synthetic (man-made) allotetraploids of Arabidopsis exhibit rapid changes in gene regulation, including gene silencing. These regulatory abnormalities could derive from ploidy changes and/or incompatible interactions between parental genomes, although comparison of auto- and allopolyploids suggests that intergenomic incompatibilities play the major role. Models to explain intergenomic incompatibilities incorporate both genetic and epigenetic mechanisms. In one model, the activation of heterochromatic transposons (McClintock's genomic shock) may lead to widespread perturbation of gene expression, perhaps by a silencing interaction between activated transposons and euchromatic genes. Qualitatively similar responses, of lesser intensity, may occur in intraspecific hybrids. Therefore, insight into genome function gained from the study of allopolyploidy may be applicable to hybrids of any type and may even elucidate positive interactions, such as those responsible for hybrid vigor.     

The genome in space and time: Does form always follow function?

Recent systematic studies using newly developed genomic approaches have revealed common mechanisms and principles that underpin the spatial organization of eukaryotic genomes and allow them to respond and adapt to diverse functional demands. Genomes harbor, interpret, and propagate genetic and epigenetic information, and the three-dimensional (3D) organization of genomes in the nucleus should be intrinsically linked to their biological functions. However, our understanding of the mechanisms underlying both the topological organization of genomes and the various nuclear processes is still largely incomplete. In this essay, we focus on the functional relevance as well as the biophysical properties of common organizational themes in genomes (e.g. looping, clustering, compartmentalization, and dynamics), and examine the interconnection between genome structure and function from this angle. Present evidence supports the idea that, in general, genome architecture reflects and influences genome function, and is relatively stable. However, the answer as to whether genome architecture is a hallmark of cell identity remains elusive.     

The gene-silencing efficiency of siRNA is strongly dependent on the local structure of mRNA at the targeted region

The gene-silencing effect of short interfering RNA (siRNA) is known to vary strongly with the targeted position of the mRNA. A number of hypotheses have been suggested to explain this phenomenon. We would like to test if this positional effect is mainly due to the secondary structure of the mRNA at the target site. We proposed that this structural factor can be characterized by a single parameter called "the hydrogen bond (H-b) index," which represents the average number of hydrogen bonds formed between nucleotides in the target region and the rest of the mRNA. This index can be determined using a computational approach. We tested the correlation between the H-b index and the gene-silencing effects on three genes (Bcl-2, hTF, and cyclin B1) using a variety of siRNAs. We found that the gene-silencing effect is inversely dependent on the H-b index, indicating that the local mRNA structure at the targeted site is the main cause of the positional effect. Based on this finding, we suggest that the H-b index can be a useful guideline for future siRNA design.     

Geminivirus-based vectors for gene silencing in Arabidopsis

Gene silencing, or RNA interference, is a powerful tool for elucidating gene function in Caenorhabditis elegans and Drosophila melanogaster. The vast genetic, developmental and sequence information available for Arabidopsis thaliana makes this an attractive organism in which to develop reliable gene-silencing tools for the plant world. We have developed a system based on the bipartite geminivirus cabbage leaf curl virus (CbLCV) that allows silencing of endogenous genes singly or in combinations in Arabidopsis. Two vectors were tested: a gene-replacement vector derived from the A component; and an insertion vector derived from the B component. Extensive silencing was produced in new growth from the A component vectors, while only minimal silencing and symptoms were seen in the B component vector. Two endogenous genes were silenced simultaneously from the A component vector and silencing of the genes was maintained throughout new growth. Because the CbLCV vectors are DNA vectors they can be inoculated directly from plasmid DNA. Introduction of these vectors into intact plants bypasses transformation and extends the kinds of silencing studies that can be carried out in Arabidopsis.     

The evolutionary mechanism of cancer

Identification of the general molecular mechanism of cancer is the Holy Grail of cancer research. Since cancer is believed to be caused by a sequential accumulation of cancer gene mutations, the identification, characterization, and targeting of common genetic alterations and their defined pathways have dominated the field for decades. Despite the impressive data accumulated from studies of gene mutations, epigenetic dysregulation, and pathway alterations, an overwhelming amount of diverse molecular information has offered limited understanding of the general mechanisms of cancer. To solve this paradox, the newly established genome theory is introduced here describing how somatic cells evolve within individual patients. The evolutionary mechanism of cancer is characterized using only three key components of somatic cell evolution that include increased system dynamics induced by stress, elevated genetic and epigenetic heterogeneity, and genome alteration mediated natural selection. Cancer progression represents a macro‐evolutionary process where karyotype change or genome replacement plays the key dominant role. Furthermore, the recently identified relationship between the evolutionary mechanism and a large number of diverse individual molecular mechanisms is discussed. The total sum of all the individual molecular mechanisms is equal to the evolutionary mechanism of cancer. Individual molecular mechanisms including all the molecular mechanisms described to date are stochastically selected and unpredictable and are therefore clinically impractical. Recognizing the fundamental importance of the underlying basis of the evolutionary mechanism of cancer mandates the development of new strategies in cancer research. J. Cell. Biochem. 109: 1072–1084, 2010. © 2010 Wiley‐Liss, Inc.     

Initial evidence of functional siRNA machinery in dinoflagellates

Dinoflagellates are a major group of protists widely distributed in the aquatic environments. Many species in this lineage are able to form harmful algal blooms (HAB), some even producing toxins, making this phylum the most important contributors of HAB in the marine ecosystem. Despite the ecological importance, the molecular mechanisms underpinning the basic biology and HAB formation of dinoflagellates are poorly understood. While the high-throughput sequencing studies have documented a large and growing number of genes in dinoflagellates, their functions remained to be experimentally proven using a functional genetic tool. Unfortunately, no such tool is yet available. This study was aimed to adopt the RNA interference (RNAi) gene-silencing tool for dinoflagellate research, and to investigate the potential effects of RNAi-based silencing of proton-pump rhodopsin and CO₂-fixing enzyme Rubisco encoding genes in dinoflagellates. It was found that RNAi treatment caused a significant decrease in growth rate in both species. Compared with the non- endogenous target (GFP-siRNA) and the blank control, RNAi treatments also suppressed the expression of the target genes. These results constitute the first experimental evidence of the existence and operation of siRNA in two species of dinoflagellates, present initial evidence that dinoflagellate rhodopsins are functional as a supplemental energy acquisition mechanism, and provide technical information for future functional genetic research on dinoflagellates.     

The revolution of the biology of the genome

Sequence data of entire eukaryotic genomes and their detailed comparison have provided new evidence on genome evolution. The major mechanisms involved in the increase of genome sizes are polyploidization and gene duplication.Subsequent gene silencing or mutations, preferentially in regulatory sequences of genes, modify the genome and permit the development of genes with new properties. Mechanisms such as lateral gene transfer, exon shuffling or the creation of new genes by transposition contribute to the evolution of a genome, but remain of relatively restricted relevance.Mechanisms to decrease genome sizes and, in particular, to remove specific DNA sequences, such as blocks of satellite DNAs, appear to involve the action of RNA interference (RNAi). RNAi mechanisms have been proven to be involved in chromatin packaging related with gene inactivation as well as in DNA excision during the macronucleus development in ciliates.     

Whole genome plasticity in pathogenic bacteria

The exploitation of bacterial genome sequences has so far provided a wealth of new general information about the genetic diversity of bacteria, such as that of many pathogens. Comparative genomics uncovered many genome variations in closely related bacteria and revealed basic principles involved in bacterial diversification, improving our knowledge of the evolution of bacterial pathogens. A correlation between metabolic versatility and genome size has become evident. The degenerated life styles of obligate intracellular pathogens correlate with significantly reduced genome sizes, a phenomenon that has been termed "evolution by reduction". These mechanisms can permanently alter bacterial genotypes and result in adaptation to their environment by genome optimization. In this review, we summarize the recent results of genome-wide approaches to studying the genetic diversity of pathogenic bacteria that indicate that the acquisition of DNA and the loss of genetic information are two important mechanisms that contribute to strain-specific differences in genome content.