Chandrakanth Emani

Developmental and tissue-specific expression of CaMV 35S promoter in cotton as revealed by GFP

The CaMV 35S promoter is the most commonly used promoter for driving transgene expression in plants. Though it is presumed to be a constitutive promoter, some reports suggest that it is not expressed in all cell types. In addition, the information available on its expression profile in all possible cell and tissue types and during early stages of development is incomplete. We present here a detailed expression profile of this promoter investigated using the green fluorescent protein (GFP) gene as a reporter system in cotton during embryo development, and in all the vegetative and floral cell and tissue types. GFP expression was not detected during the early stages of embryogenesis. The first perceptible GFP expression was observed in a small area at the junction of hypocotyl and cotyledons in embryos at around 13 days after anthesis. The GFP fluorescence progressively became stronger and expanded throughout the cotyledon and hypocotyl as embryo development advanced. After germination, varying levels of promoter activity were observed in all cell and tissue types in the hypocotyl, cotyledon, stem, leaf, petiole, and root. The promoter was also expressed in all floral parts. Although cotton pollen exhibited a low level of greenish autofluorescence, it was possible to discern GFP-dependent fluorescence in some of the pollen from all the T0 plants examined. Developing cotton fibers also exhibited GFP fluorescence suggesting that the 35S promoter was active in these specialized epidermal cells. Thus, we show that the expression of the 35S promoter was developmentally regulated during embryogenesis and that beyond a certain stage during embryogenesis, the promoter was expressed in most cell and tissue types in cotton albeit at different levels.

Transgene silencing and reactivation in sorghum

Amongst the important cereals, sorghum has been the most recalcitrant to genetic transformation. There are a few reports on sorghum transformation and the majority of these have reported silencing of one of the transgenes. We regenerated plants from two independent transgenic sorghum callus lines that were cotransformed with Ubi promoter:bar and Act1-D promoter:gusA gene constructs using the particle bombardment method. Southern analyses indicated integration of multiple copies of both the transgenes. T0 plants were found to express the bar gene. The gusA gene, however, was silenced. It was possible to activate gusA gene expression in T1 seedlings and in calli derived from immature T1 and T2 embryos by 5-azacytidine (azaC) treatment. In certain cases, spontaneous expression of the gusA gene was observed in T1 and T2 immature embryo-derived calli. Expression of the bar gene, as analyzed by Basta™ tolerance and Phosphinothricin acetyltransferase (PAT) assays, was detected in T0, T1 and T2 plantlets; however, the expression was reduced in the T2 progeny obtained from a homozygous T1 parent. PAT activity was also lower in the immature embryo-derived T2 calli from the same homozygous T1 parent. Again, culture on azaC increased the level of PAT activity in these calli. Moreover, in a separate set of stable transformation experiments, it was possible to recover a much higher than usual number of gusA gene expressing transgenic calli by growing the bombarded tissues in the presence of azaC. Taken together, these results suggest that methylation-based silencing is frequent in sorghum and probably responsible for several cases of transgene inactivation reported earlier for this crop.

Sugarcane DIRIGENT and O-METHYLTRANSFERASE promoters confer stem-regulated gene expression in diverse monocots.

Transcription profiling analysis identified Saccharum hybrid DIRIGENT (SHDIR16) and Ο-METHYLTRANSFERASE (SHOMT), putative defense and fiber biosynthesis-related genes that are highly expressed in the stem of sugarcane, a major sucrose accumulator and biomass producer. Promoters (Pro) of these genes were isolated and fused to the β-glucuronidase (GUS) reporter gene. Transient and stable transgene expression analyses showed that both ProDIR16:GUS and ProOMT:GUS retain the expression characteristics of their respective endogenous genes in sugarcane and function in orthologous monocot species, including rice, maize and sorghum. Furthermore, both promoters conferred stem-regulated expression, which was further enhanced in the stem and induced in the leaf and root by salicylic acid, jasmonic acid and methyl jasmonate, key regulators of biotic and abiotic stresses. ProDIR16 and ProOMT will enable functional gene analysis in monocots, and will facilitate engineering monocots for improved carbon metabolism, enhanced stress tolerance and bioenergy production.

Phaseolin: Structure and Evolution

Phaseolin is the salt-soluble glycoprotein or the group of polypeptides of the French bean (Phaseolus vulgaris L.) that account for some 50% of the total protein in mature bean seeds. It was one of the first plant proteins to be trans- lated in vitro from mRNA and one of the first plant genes isolated. It was also the first developmentally regulated plant gene to be expressed in a heterologous plant species through Agrobacterium-mediated transformation. Studies on phaseo- lin have provided insight to many aspects of plant protein synthesis, from fundamental molecular mechanisms to practical goals such as the improvement of the French beans nutritional quality. The present review is a comprehensive account of the structural and functional features of phaseolin that have implications regarding its evolution. Additionally, future di- rections in phaseolin evolutionary studies and suggestions regarding effective and safe biotechnological approaches for the nutritional improvement of French bean seed are outlined.

Genome Editing in Plants: An Overview of Tools and Applications

The emergence of genome manipulation methods promises a real revolution in biotechnology and genetic engineering. Targeted editing of the genomes of living organisms not only permits investigations into the understanding of the fundamental basis of biological systems but also allows addressing a wide range of goals towards improving productivity and quality of crops. This includes the creation of plants with valuable compositional properties and with traits that confer resistance to various biotic and abiotic stresses. During the past few years, several novel genome editing systems have been developed; these include zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and clustered regularly interspaced short palindromic repeats/Cas9 (CRISPR/Cas9). These exciting new methods, briefly reviewed herein, have proved themselves as effective and reliable tools for the genetic improvement of plants.

Using the “DNA Story” to Inculcate a Scientific Thought Process in the Classroom

ABSTRACT Episodic history of major scientific events can be an effective tool for sharing the excitement and eureka moments of scientific revolutions, especially in classrooms with both science and nonscience majors. By introducing the principal characters and the timeline involved in a scientific discovery, teachers can inculcate a scientific thought process in the students. This may in turn lead to a classroom environment in which students can develop analytical skills that are required to understand the basic concepts and the underlying characteristics of a scientific theory, instead of blindly accepting textbook facts.

Transgenic Cotton for Agronomical Useful Traits

Cotton (Gossypium spp.) is an important crop known for its commercial significance both as a fiber and oil yielding cultivar grown in over eighty countries around the world. The crop’s economic importance underlined by its significance in textile industry makes it all the more important for agronomists and crop researchers to strategize novel approaches to overcome challenges in the form of abiotic and biotic stresses such as pests, pathogens, weeds, and more recently environmental challenges in the form of climate change. Conventional breeding technology made significant in roads into developing novel varieties with improved fiber, enhanced heat tolerance and high yields, but significant challenges remain in the areas of pathogen and insect resistance. Transgenesis or genetic engineering was an ideal solution to these challenges with its ability to introduce diverse agronomical important genes from various biological species, and over 80 % of cotton grown currently employs this technology. The present review is a comprehensive account of the historical progress made in the area of transgenic cotton both in terms of the evolution of the methodologies of tissue culture and genetic transformation, and lessons learned from conventional breeding in identifying agronomical important genes to improve cotton production.

Metabolic Engineering of Pathways and Gene Discovery

Humans have been manipulating the genetic information of plants throughout the history of agriculture. In this respect, every new plant variety or animal race is a result of the introduction of novel metabolic changes. This process has been slowly advancing for millennia. However, with the discovery of biochemical pathways and later with the introduction of gene manipulation techniques in 1970s, the pace greatly speeded up. Already in the mid-1980s, many of the compounds and enzymes participating in metabolic pathways were linked to their cloned genes, which can then be used for engineering the plant metabolism. Soon, novel products from plants appeared including, vaccines and other pharmaceuticals, plastics, and proteins that may render certain plants as effective tools for environmental decontamination. These products were a result of the manipulation of plant endogenous biochemical pathways and thus the novel field of science-metabolic engineering was born. Metabolic engineering can be defined as the targeted and purposeful modification of metabolic pathways in an organism for the improved use of cellular pathways for chemical transformation, energy transduction, and macromolecular synthesis or breakdown, potentially benefiting the society by producing biological substitutes for toxic chemicals, increasing agricultural production, improving industrial fermentation processes, producing completely new compounds, or by understanding the molecular mechanism underlying medical conditions in order to develop new cures (Kurnaz 2005).

Role of MicroRNAs and small RNAs in regulation of developmental processes and agronomic traits in Gossypium species

Small RNAs (sRNAs) are short, non-coding, 17-24 nucleotides long RNA molecules that play vital roles in regulating gene expression in every known organism investigated to date including cotton (Gossypium ssp.). These tiny RNA molecules target diverse categories of genes from different bioliogical and metabolic processes and have been reported in the three domains of life. Small RNAs, including miRNAs, are involved in ovule and fiber development, biotic and abiotic stresses, fertility, and other biochemical processes in cotton species. Also, sRNAs are the critical components in RNA interference pathway. In this article, we have reviewed the research efforts related to the isolation and characterization of miRNAs using molecular and genomic approaches. The progress made in understanding the functional roles of miRNAs in regulation, alteration, and inactivation of fundamental plant processes and traits of importance in cotton are presented here.

Proceedings of the 15th Annual UT-KBRIN Bioinformatics Summit 2016

Table of contentsI1 Proceedings of the Fifteenth Annual UT- KBRIN Bioinformatics Summit 2016Eric C. Rouchka, Julia H. Chariker, Benjamin J. Harrison, Juw Won ParkP1 CC-PROMISE: Projection onto the Most Interesting Statistical Evidence (PROMISE) with Canonical Correlation to integrate gene expression and methylation data with multiple pharmacologic and clinical endpointsXueyuan Cao, Stanley Pounds, Susana Raimondi, James Downing, Raul Ribeiro, Jeffery Rubnitz, Jatinder LambaP2 Integration of microRNA-mRNA interaction networks with gene expression data to increase experimental powerBernie J Daigle, Jr.P3 Designing and writing software for in silico subtractive hybridization of large eukaryotic genomesDeborah Burgess, Stephanie Gehrlich, John C CarmenP4 Tracking the molecular evolution of Pax geneNicholas Johnson; Chandrakanth EmaniP5 Identifying genetic differences in thermally dimorphic and state specific fungi using in silico genomic comparisonStephanie Gehrlich, Deborah Burgess, John C CarmenP6 Identification of conserved genomic regions and variation therein amongst Cetartiodactyla species using next generation sequencingKalpani De Silva, Michael P Heaton, Theodore S KalbfleischP7 Mining physiological data to identify patients with similar medical events and phenotypesTeeradache Viangteeravat, Rahul Mudunuri, Oluwaseun Ajayi, Fatih Şen, Eunice Y HuangP8 Smart brief for home health monitoringMohammad Mohebbi, Luaire Florian, Douglas J Jackson, John F NaberP9 Side-effect term matching for computational adverse drug reaction predictionsAKM Sabbir, Sally R EllingsonP10 Enrichment vs robustness: A comparison of transcriptomic data clustering metricsYuping Lu, Charles A Phillips, Michael A LangstonP11 Deep neural networks for transcriptome-based cancer classificationRahul K Sevakula, Raghuveer Thirukovalluru, Nishchal K. Verma, Yan CuiP12 Motif discovery using K-means clusteringMohammed Sayed, Juw Won ParkP13 Large scale discovery of active enhancers from nascent RNA sequencingJing Wang, Qi Liu, Yu ShyrP14 Computationally characterizing genomic pipelines and benchmarking results using GATK best practices on the high performance computing cluster at the University of KentuckyXiaofei Zhang, Sally R EllingsonP15 Development of approaches enabling the identification of abnormal gene expression from RNA-Seq in personalized oncologyNaresh Prodduturi, Gavin R Oliver, Diane Grill, Jie Na, Jeanette Eckel-Passow, Eric W KleeP16 Processing RNA-Seq data of plants infected with coffee ringspot virusMichael M Goodin, Mark Farman, Harrison Inocencio, Chanyong Jang, Jerzy W Jaromczyk, Neil Moore, Kelly SovacoolP17 Comparative transcriptomics of three Acinetobacter baumanii clinical isolates with different antibiotic resistance patternsLeon Dent, Mike Izban, Sammed Mandape, Shruti Sakhare, Siddharth Pratap, Dana MarshallP18 Metagenomic assessment of possible microbial contamination in the equine reference genome assemblyM Scotty DePriest, James N MacLeod, Theodore S KalbfleischP19 Molecular evolution of cancer driver genesChandrakanth Emani, Hanady Adam, Ethan Blandford, Joel Campbell, Joshua Castlen, Brittany Dixon, Ginger Gilbert, Aaron Hall, Philip Kreisle, Jessica Lasher, Bethany Oakes, Allison Speer, Maximilian ValentineP20 Biorepository Laboratory Information Management SystemNaga Satya V Rao Nagisetty, Rony Jose, Teeradache Viangteeravat, Robert Rooney, David Hains