Hindawi Publishing Corporation International Journal of Plant Genomics
stresses (cold, and sulfate starvation); others
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stresses (cold, and sulfate starvation); others (protein amino acid phosphorylation; protein folding, and biological processes unknown) Biosynthesis (alkaloids, folic acid and its derivatives, trehalose, and tRNA processing); transport (metal ions, and protein import into nucleus); gene regulation (DNA methylation, nuclear mRNA splicing; posttranscriptional gene silencing, and transcription factors); response to biotic and abiotic stresses (oxidative stresses); others (protein modification process, and biological processes unknown) Biosynthesis (glycogenin); metabolism (glycolysis, ATP-dependent proteolysis, and protein ubiquitination); transport (cation and intracellular protein); cell growth and organogenesis (ethylene mediated unidimensional cell growth); gene regulation (chromatinmodification, RNA processing, and transcription factors); response to biotic/abiotic stresses (response to salt stress); signal transduction; others (gravitropism, and biological processes unknown) Biosynthesis (ATP, lignins, translation, and ribosome biogenesis and assembly); metabolism (amino acids; lipids, prolines, steroids, and proteolysis); transport (calcium ion, cations, electrons, and potassium ion); cell growth and organogenesis (embryonic development ending in seed dormancy, fruit development, microtubule-based process, unidimensional cell growth, cellulose and pectin-containing cell wall modification, tubulin folding, cytokinesis, and cellulose and pectin-containing cell wall loosening); gene regulation (transcription factors); photomorphogenesis (response to red or far red light); respond to biotic/abiotic
mismatch repair); others (regulation of GTPase activity, vesicle docking during exocytosis, circadian rhythm, and biological processes unknown)
lipid glycosylation, proteins, fatty acid beta-oxidation, proteolysis, photorespiration); transport (protein import into peroxisome matrix); cell growth and organogenesis (embryonic development ending in seed dormancy, and peroxisome organization and biogenesis); gene regulation (DNA methylation, genetic imprinting, and transcription factor); response to biotic/abiotic stresses (defense response); signal transduction; others (protein folding, protein amino acid glycosylation, and biological processes unknown) Metabolism (carbohydrates); transport; cell growth and organogenesis (flower development and organogenesis); gene regulation (transcription factors); response to biotic/abiotic stresses (temperature stimulus);
and biological processes unknown) Biosynthesis (histidine, tryptophan, and proteins); transport (anions, and electron); cell growth and organogenesis (root hair elongation); gene regulation (transcription factors); others (biological processes unknown Biosynthesis (acetyl-CoA, ethylene, fatty acids, and lipid A); metabolism (D-ribose metabolic process, and glycolysis,); transport (electron, mitochondrial, and vesicle-mediated); cell growth and organogenesis (cell proliferation, actin filament-based process, structural constituent of cytoskeleton, microtubule-based process, and vacuole organization and biogenesis); gene regulation (transcription factors); photomorphogenesis (red, far-red, light signaling pathway, response to red light, negative regulation of photomorphogenesis, response to far red light, short-day photoperiodism, and negative regulation of flower development); response to biotic/abiotic stresses (response to cold); signal transduction (small GTPase mediated signal transduction); DNA biogenesis (DNA repair); others (N-terminal protein myristoylation, and biological processes unknown)
serine-isocitrate lyase pathway, glyceraldehyde-3-phosphate catabrolism, aerobic glycerol catabolism, anaerobic glycolysis, non-phosphorylated glucose catabolism, acetate fermentation, glucose catabolism to D-lactate and ethanol, glucose catabolism to butanediol, glucose catabolism to lactate and acetate, protolysis, ATP-dependent proteolysis, and medium-chain fatty acid); transport (amino acids, auxin polar, electrons, and oligopeptides); cell growth and
development, pollen wall formation, and regulation of progression through cell cycle); gene regulation (transcription factor); response to phytohormones (auxin and ethylene stimuli); response to biotic/abiotic stresses (defense response, light stimulus, salt stress and wounding); signal transduction (salicylic acid mediated signaling pathway); others (cellular calcium ion homeostasis; protein amino acid dephosphorylation and protein amino acid phosphorylation; protein folding; protein modification process, N-terminal protein myristoylation, positive gravitropism, and biological processes unknown)
aromatic compounds, carbohydrates, glucans, phosphatidylcholine, nitrogen compounds, nucleobases, nucleosides, nucleotides and nucleic acids, and proteolysis); transport (amino acids, sulfates, and vesicle-mediated); cell growth and organogenesis (shoot development, cell adhesion, cellulose and pectin-containing cell wall biogenesis, microtubule cytoskeleton organization and biogenesis, pollen tube growth, and cell morphogenesis); photomorphogenesis (photoperiodism, response to light stimulus, and regulation of flower development); response to phytohormones (auxin and cytokinin stimuli); response to biotic/abiotic stresses (defense response to nematode); signal
system); DNA biogenesis (DNA repair and replication); others (protein amino acid phosphorylation, biological processes unknown) 2 1 4 9 8 3 10 6 7 0 5 Figure 7: Annotation of biological processes targeted by abundant copy ( >5 copies) candidate siRNAs of developing ovules in cotton. To better visualize the specific and overlapping putatively targeted proteins at 0 to 10 DPA ovules, Cytoscape [ 43 ] was used to generate genetic interaction networks of putative targets at di fferent DPA stages of ovule, where each node (DPA) and its edges (targeted proteins) were colored. The interaction networks were depicted using Cytoscape’s “spring embedded layout algorithm” for both full protein target dataset and protein groups targeted by only abundant copy candidate siRNAs, importing the “simple interaction format (SIF) files” into Cytoscape. SIF files were created based on specific and overlapping target protein information for 0–10 DPA ovule stages [ 33 ]. (3) Select RNA fragment(s) to be purified and cut it (them) from the gel as shown in Figure 8 . (4) Place the gel slice in a 1.5 mL tube and crush with a glass rod. (Note: we have had very good results using the 1.5 mL tubes and disposable pestles from Kontes Glass Company.) (5) Add 200 μL IDT sterile, nuclease-free water and continue to crush the gel into a fine slurry. Place the tube at 70 ◦ C for 10 minutes. (6) Following manufacturer’s recommendations, prepare a Performa DTR column for each gel slice. (7) Vortex the gel slurry, transfer the entire volume onto the column and spin at 3000 rpm for 3 minutes. (8) Discard the DTR column. (9) Add 3 μL 10 mg/ml glycogen, 25 μL of 3M NaOAc (pH5.2), and 900 μL ice cold 100% EtOH to the eluent. Mix by inversion and place at − 80
C for 20 minutes.
(10) Spin tubes at full speed ( ≥ 10 000 rpm) for 10 minutes to pellet the RNA. Pour o ff the supernatant and dry the pellet. (11) Proceed to next procedure/application (e.g., miRCat protocol). This protocol successfully removes the Urea and other salts with substantially less loss of RNA than is seen with conventional crush and soak methods fol- lowed by NAP-5 column desalting or by dialysis meth- ods. Detail list of small RNA cloning products and protocol for miRCat can be found from IDT prod- uct manual at ( http://www.idtdna.com/Support/Technical/ TechnicalBulletinPDF/miRCat User Guide.pdf ).
International Journal of Plant Genomics 11 5.8s rRNA tRNAs Gel Slice 21-mer miSPIKE™ control RNA Total RNA 5s rRNA
Figure 8 Acknowledgments Cotton small RNA characterization research was funded by Academy of Sciences of Uzbekistan under research Grant 4F-P-149. The authors are grateful to the United States Department of Agriculture/Agricultural Research Services (USDA/ARS)—Former Soviet Union (FSU) Scientific Coop- eration Program, O ffice of International Research Programs, USDA/ARS for financial support of cotton genomics research in Uzbekistan. The authors thank anonymous reviewers of the manuscript for valuable suggestions. References [1] C. Napoli, C. Lemieux, and R. Jorgensen, “Introduction of a chimeric chalcone synthase gene into petunia results in reversible co-suppression of homologous genes in trans,” The Plant Cell, vol. 2, no. 4, pp. 279–289, 1990. [2] A. R. van der Krol, L. A. Mur, M. Beld, J. N. M. Mol, and A. R. Stuitje, “Flavonoid genes in petunia: addition of a limited number of gene copies may lead to a suppression of gene expression,” The Plant Cell, vol. 2, no. 4, pp. 291–299, 1990. [3] N. Romano and G. Macino, “Quelling: transient inactivation of gene expression in Neurospora crassa by transformation with homologous sequences,” Molecular Microbiology, vol. 6, no. 22, pp. 3343–3353, 1992. [4] C. Cogoni and G. Macino, “Isolation of quelling-defective (qde) mutants impaired in posttranscriptional transgene- induced gene silencing in Neurospora crassa,” Proceedings of the National Academy of Sciences of the United States of America, vol. 94, no. 19, pp. 10233–10238, 1997. [5] A. Fire, S. Xu, M. K. Montgomery, S. A. Kostas, S. E. Driver, and C. C. Mello, “Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans,” Nature, vol. 391, no. 6669, pp. 806–811, 1998. [6] G. Meister and T. Tuschl, “Mechanisms of gene silencing by double-stranded RNA,” Nature, vol. 431, no. 7006, pp. 343– 349, 2004. [7] R. C. Lee, R. L. Feinbaum, and V. Ambros, “The C. elegans heterochronic gene lin-4 encodes small RNAs with antisense complementarity to lin-14,” Cell, vol. 75, no. 5, pp. 843–854, 1993. [8] B. Wightman, I. Ha, and G. Ruvkun, “Posttranscriptional regulation of the heterochronic gene lin-14 by lin-4 mediates temporal pattern formation in C. elegans,” Cell, vol. 75, no. 5, pp. 855–862, 1993. [9] M. Lagos-Quintana, R. Rauhut, W. Lendeckel, and T. Tuschl, “Identification of novel genes coding for small expressed RNAs,” Science, vol. 294, no. 5543, pp. 853–858, 2001. [10] N. C. Lau, L. P. Lim, E. G. Weinstein, and D. P. Bartel, “An abundant class of tiny RNAs with probable regulatory roles in Caenorhabditis elegans,” Science, vol. 294, no. 5543, pp. 858– 862, 2001. [11] R. C. Lee and V. Ambros, “An extensive class of small RNAs in
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