番木瓜中6个BG生物合成相关基因荧光定量分析

异硫氰酸苄酯(benzyl isothiocyanate,BITC)是医学界公认的具有防癌抗癌活性的成分,对多种癌症具有很好的作用.BG是BITC的前体物质,可以在芥子酶的作用下水解生成BITC.研究表明,BG在热带水果番木瓜中含量丰富.本研究在克隆了番木瓜中6个相关基因和测定了BG在番木瓜不同组织中含量的基础上,采用实时荧光定量PCR技术,对6个基因在番木瓜不同组织中的表达量进行测定,并与BG的含量进行比较,考察其相关性.结果表明各基因的表达量相对较高时BG的含量也较高,即6个基因在番木瓜中的表达量与BG的含量存在相关性.我们推测,这6个基因可能是番木瓜中BG生物合成相关基因.本文结果为进一步研究基因的功能提供了理论依据.     

A protocol for efficient transformation and regeneration of Carica papaya L.

Summary A reproducible and effective biolistic method for transforming papaya (Carica papaya L.) was developed with a transformation-regeneration system that targeted a thin layer of embryogenic tissue. The key factors in this protocol included: 1) spreading of young somatic embryo tissue that arose directly from excised immature zygotic embryos, followed by another spreading of the actively growing embryogenic tissue 3 d before biolistic transformation; 2) removal of kanamycin selection from all subsequent steps after kanamycin-resistant clusters were first isolated from induction media containing kanamycin; 3) transfer of embryos with finger-like extensions to maturation medium; and 4) transferring explants from germination to the root development medium only after the explants had elongating root initials, had at least two green true leaves, and were about 0.5 to 1.0 cm tall. A total of 83 transgenic papaya lines expressing the nontranslatable coat protein gene of papaya ringspot virus (PRSV) were obtained from somatic embryo clusters that originated from 63 immature zygotic embryos. The transformation efficiency was very high: 100% of the bombarded plates produced transgenic plants. This also represents an average of 55 transgenic lines per gram fresh weight, or 1.3 transgenic lines per embryo cluster that was spread. We validated this procedure in our laboratory by visiting researchers who did four independent projects to transform seven papaya cultivars with coat protein gene constructs of PRSV strains from four different countries. The method is described in detail and should be useful for the routine transformation and regeneration of papaya. Based in part on a presentation at the 1997 SIVB Congress on In Vitro Biology held in Washington, DC, June 14–18, 1997.     

A small-scale screening technique for evaluating fungicides against Phytophthora palmivora pod rot of cocoa

Plots of clonal Trinitario cocoa were sprayed with fungicides in the field. Ripe pods were harvested from the trial plots and artificially inoculated with Phytophthora palmivora using various techniques, the most suitable of which proved to be the zoospore spot method. Percentage infection results from the spot inoculation tests agreed well with the field percentage infection results and it was concluded that field spraying followed by zoospore spot inoculation of detached pods is a suitable preliminary screening technique for fungicides. Three systemic fungicides metalaxyl, aluminium tris (ethyl phosphonate) and DPX-3217 in a mixture with captafol and copper oxychloride were compared with the protectant cuprous oxide. Metalaxyl was found to be the most effective of the systemic fungicides and when applied at the rate of 0·44 g ai/ha every 8 wk it gave a similar reduction in percentage infection to 4 weekly applications of cuprous oxide at the rate of 4·4 kg Cu⁺⁺/ha during the wetter part of the year when pod rot is most severe.     

A General Model for Multilocus Epistatic Interactions in Case-Control Studies

Background

Epistasis, i.e., the interaction of alleles at different loci, is thought to play a central role in the formation and progression of complex diseases. The complexity of disease expression should arise from a complex network of epistatic interactions involving multiple genes.

Methodology

We develop a general model for testing high-order epistatic interactions for a complex disease in a case-control study. We incorporate the quantitative genetic theory of high-order epistasis into the setting of cases and controls sampled from a natural population. The new model allows the identification and testing of epistasis and its various genetic components.

Conclusions

Simulation studies were used to examine the power and false positive rates of the model under different sampling strategies. The model was used to detect epistasis in a case-control study of inflammatory bowel disease, in which five SNPs at a candidate gene were typed, leading to the identification of a significant three-locus epistasis.     

Phenolic Content in Male and Female Carica papaya: A Possible Physiological Marker for Sex Identification of Vegetative Seedlings

The phenolic composition was determined in the leaves, petioles and bark tissues of male and female plants of two papaya cultivars. The same kind of phenolics were isolated from the male and female plants. However, a marked difference was observed between the plant organs of different cultivars. The important free and bound phenolics extracted after acidic and alkaline hydrolysis were caffeic acid, gentisic acid, m-coumaric acid, p-coumaric acid, salicylic acid and quercetin. Four phenolic compounds were not identified. The amounts of free, acid-hydrolysable and alkali-hydrolysable phenolic compounds were considerably higher in male plants.     

The Torpedo Electrocyte: A Model System for the Study of Receptor-Cytoskeleton Interactions

Abstract

We have used the electrocyte of Torpedo electric organ as a model system for the study of AchR stabilization in the post-synaptic membrane. Attention was focused on membrane cytoskeleton interactions in particular on a peripheral protein of 43 KD that is believed to participate in AchR immobilization.

Using immunocytochemical methods, we have shown that the cortical skeleton in Torpedo electrocyte displays a local differentiation proper for each specialized domain of the plasma membrane. In the postsynaptic membrane, characterized by an accumulation and a geometrical organization of the receptors in the plane of the membrane, the 43 KD protein participates in a submembraneous coating or “postsynaptic densities” that strictly codistribute with the AchR. The 43 KD protein might also account for the anchoring of intermediate-sized filaments.

The organization of the postsynaptic domain appears readily different from that of the non-innervated one where the membrane folds are maintained by a cortical meshwork of cytoskeletal proteins such as ankyrin, spectrin and oligomeric actin.

In conclusion, the asymmetrical organization of the cortical skeleton in the electrocyte offers a unique opportunity for the study of the specific aspects of membrane-skeleton interactions that take place in the postsynaptic domain.     

Development of a Gene-Centered SSR Atlas as a Resource for Papaya (Carica papaya) Marker-Assisted Selection and Population Genetic Studies

Carica papaya (papaya) is an economically important tropical fruit. Molecular marker-assisted selection is an inexpensive and reliable tool that has been widely used to improve fruit quality traits and resistance against diseases. In the present study we report the development and validation of an atlas of papaya simple sequence repeat (SSR) markers. We integrated gene predictions and functional annotations to provide a gene-centered perspective for marker-assisted selection studies. Our atlas comprises 160,318 SSRs, from which 21,231 were located in genic regions (i.e. inside exons, exon-intron junctions or introns). A total of 116,453 (72.6%) of all identified repeats were successfully mapped to one of the nine papaya linkage groups. Primer pairs were designed for markers from 9,594 genes (34.5% of the papaya gene complement). Using papaya-tomato orthology assessments, we assembled a list of 300 genes (comprising 785 SSRs) potentially involved in fruit ripening. We validated our atlas by screening 73 SSR markers (including 25 fruit ripening genes), achieving 100% amplification rate and uncovering 26% polymorphism rate between the parental genotypes (Sekati and JS12). The SSR atlas presented here is the first comprehensive gene-centered collection of annotated and genome positioned papaya SSRs. These features combined with thousands of high-quality primer pairs make the atlas an important resource for the papaya research community.     

Diversity of Interactions: A Metric for Studies of Biodiversity

Multitrophic interactions play key roles in the origin and maintenance of species diversity, and the study of these interactions has contributed to important theoretical advances in ecology and evolutionary biology. Nevertheless, most biodiversity inventories focus on static species lists, and prominent theories of diversity still ignore trophic interactions. The lack of a simple interaction metric that is analogous to species richness is one reason why diversity of interactions is not examined as a response or predictor variable in diversity studies. Using plant–herbivore–enemy trophic chains as an example, we develop a simple metric of diversity in which richness, diversity indices (e.g., Simpson's 1/D), and rarefaction diversity are calculated with links as the basic unit rather than species. Interactions include all two-link (herbivore–plant and enemy–herbivore) and three-link (enemy–herbivore–plant) chains found in a study unit. This metric is different from other indices, such as traditional diversity measures, connectivity and interaction diversity in food-web studies, and the diversity of interaction index in behavioral studies, and it is easier to compute. Using this approach to studying diversity provides novel insight into debates about neutrality and correlations between diversity, stability, productivity, and ecosystem services.     

A Statistical Framework for Improving Genomic Annotations of Prokaryotic Essential Genes

Large-scale systematic analysis of gene essentiality is an important step closer toward unraveling the complex relationship between genotypes and phenotypes. Such analysis cannot be accomplished without unbiased and accurate annotations of essential genes. In current genomic databases, most of the essential gene annotations are derived from whole-genome transposon mutagenesis (TM), the most frequently used experimental approach for determining essential genes in microorganisms under defined conditions. However, there are substantial systematic biases associated with TM experiments. In this study, we developed a novel Poisson model–based statistical framework to simulate the TM insertion process and subsequently correct the experimental biases. We first quantitatively assessed the effects of major factors that potentially influence the accuracy of TM and subsequently incorporated relevant factors into the framework. Through iteratively optimizing parameters, we inferred the actual insertion events occurred and described each gene’s essentiality on probability measure. Evaluated by the definite mapping of essential gene profile in Escherichia coli, our model significantly improved the accuracy of original TM datasets, resulting in more accurate annotations of essential genes. Our method also showed encouraging results in improving subsaturation level TM datasets. To test our model’s broad applicability to other bacteria, we applied it to Pseudomonas aeruginosa PAO1 and Francisella tularensis novicida TM datasets. We validated our predictions by literature as well as allelic exchange experiments in PAO1. Our model was correct on six of the seven tested genes. Remarkably, among all three cases that our predictions contradicted the TM assignments, experimental validations supported our predictions. In summary, our method will be a promising tool in improving genomic annotations of essential genes and enabling large-scale explorations of gene essentiality. Our contribution is timely considering the rapidly increasing essential gene sets. A Webserver has been set up to provide convenient access to this tool. All results and source codes are available for download upon publication at http://research.cchmc.org/essentialgene/.