PLANT GENOMICS

Experiment No.1 Aim: To study the plant genome organization and evolution of Arabidopsis thaliana. Principle: The plant genome organization describes the genomic size, chromosome number, repeat sequences, how the genes are organized etc. For the genome organization and evolution the “NCBI” Databases & Tools are used. NCBI is used to predict the genome organization of any plant genome. Introduction Arabidopsis was the first plant genome to be sequenced, and is a popular tool for understanding the molecular biology of many plant traits, including flower development and light sensing. Arabidopsis thaliana (A-ra-bi-dóp-sis tha-li-á-na; thale cress, mouse-ear cress or arabidopsis) is a small flowering plant and is a popular model organism in plant biology and genetics. The advanced fields of bioinformatics and biotechnology have changed this paradigm, enabling the analysis of organisms in terms of genome organization, expression and interaction. The study of the way genes and genetic information are organized within the genome, the methods of collecting and analyzing this information and how this organization determines their biological functionality is referred to as genomics. Genomic approaches are permeating every aspect of plant biology, and since they rely on DNA-coded information, they expand molecular analyses from a single to a multispecies level. Plant genomics is reversing the previous paradigm of identifying genes behind biological functions and instead focuses on finding biological functions behind genes. It also reduces the gap between phenotype and genotype and helps to comprehend not only the isolated effect of a gene, but also the way its genetic context and the genetic networks it interacts with can modulate its activity. Requirments: Plant: Arabidopsis thaliana Database: NCBI Procedure: The genome organization and evolution is predicted by the following steps.  Open the “Genome” resource from the NCBI resources(http://www.ncbi.nlm.nih.gov/genome/)  From the custom resources of Genome select the “Plant” resource.  Select the plant Arabidopsis thaliana.  It will provide the genome organization of Arabidopsis thaliana.

Results: Genome Resource

Plant Resource: Genome Organization and Evolution: .

Arabidopsis thaliana is a diploid plant with 2n = 10 chromosomes. It is distributed throughout the world and was first reported in the sixteenth century by Johannes Thal. Genome Assembly and Annotation: Assembly and Annotation Default assembly 1 other assemblies are available Assembly Name Last sequence update Highest level of assembly Size (total bases) Number of genes Number of proteins TAIR9 19-Jun-2009 complete sequence genome 119.146.348 33. It became the first plant genome to be fully sequenced based on the fact that it has a (1) small genome of ~120 Mb with a simple structure having few repeated sequences (2) short generation time of six weeks from seed germination to seed set. brassicaceae (Cruciferae). and (3) produces large number of seeds. It is now being used as a model organism to study different aspects of plant biology. it is an invaluable resource to agriculturally important crops. The sequencing was done by an international collaboration collectively termed the Arabidopsis Genome Initiative (AGI). Though of no economic importance. which includes canola. an important source of vegetable oil. It has been used for over fifty years to study plant mutations and for classical genetic analysis.924 131 117 Chloroplast Genome Pltd 26-Mar-2010 07-Apr-2000 154.478 129 85 Arabidopsis thaliana .Arabidopsis thaliana is a small flowering plant of mustard family. particularly to members of the same family.323 35.176 Mitochondrial Genome Last record update Last sequence update Size Number of genes Number of proteins Last record update Last sequence update Size Number of genes Number of proteins 31-Jul-2008 12-Dec-2002 366.

2 Y08501.81 36. 4.263 2 NC_003071.46 36.1 18.433 96 93 79 149 5.560 3 NC_003074.9 8.323 1 1 AJ270060.7 CP002685.043 1. 14.513 134 6.1 0.8 CP002686.5 36.1 AP000423.2 5.35 1 4.7 CP002687. 14. 3.7 35.710 5 0 AE005173.43 35.228 31 92 8 4 BA000015.05 36.8 CP002688. 23. 3.1 23.140 924 1.9 CP002684.128 - 7 - AE005172.40 6 1. 3.156 1 0 AJ270058.3 6. 1 2 554 1 78 1 2.72 7 817 5 Nu Chr 4 long c arm Nu Chr 4 short c arm - - 55 - - - - 8 - - .089 MT NC_001284.1 19.67 35.730 116 5.908 4 NC_003075.2 0. 14.8 117 85 123 127 7.22 36.507 21 37 131 129 Pltd NC_000932.140 1 5 .356 5 NC_003076.98 35.83 0 2.15 36.1 30.The Arabidopsis Information Resource (TAIR) Arabidopsis assembly project Na Loc Type me Nuc Chr Nuc Chr Nuc Chr Nuc Chr Nuc Chr MT Chl Chr Chr RefSeq INSDC Size other (Mb) GC% protein rrna trna rna gene pseudogene 2 2 3 7 240 218 8.3 Arabidopsis thaliana Arabidopsis Genome Initiative Arabidopsis thaliana legacy genome sequence Loc Typ RefSe e Name q 3 - INSDC Size GC protei rrn trn other pseudogen (Mb) % n a a rna gene e 6 4.71 1 79 Nu Chr c Nu Chr c BA000014.37 44.080 832 948 - 1 NC_003070.59 36.1 26.107 5 - 10 - Nu Chr 1 bottom c arm Nu Chr c 1 top arm - .4 36. 23.9 5. 3.9 9. 5.

Conclusion: The genome organization and evolution of Arabiopsis thaliana was successfully studied. I found that Arabidopsis thaliana is a small flowering plant of mustard family having 10 chromosomes with ~ 120 Mb genome size. .

2006. which find genes in prokaryotic genomes in all six reading frames.. Jayaram B. A Physico-Chemical model for analyzing DNA sequences". Mod.0”software.Experiment No. 2 Aim of the experiment: “ To identify gene in Arabidopsis thaliana for transgenic approach in plants” Principle: For the identification of the genes in Arabidopsis thaliana I have used the “ChemGenome2. 78-85... Khurana E. 46(1). Chemgenome is an ab-intio gene prediction software. Tomer R... Agrawal P. Singhal P. The methodology follows a physico-chemical approach and has been validated on 372 prokaryotic genomes. "Decoding the design principles of amino acids and the chemical logic of protein sequences". 2.. J. Dutta S.. .0 Reference: 1. Nature Precedings. Chem. and Jayaram B. Kritee. Inf. Read more about ChemGenome Procedure: Genome: Arabidopsis thaliana chromosome1 Tool: ChemGenome 2. 2008.

Principle Outcome: Conclusion: We have successfully identifed gene in Arabidopsis thaliana which can be transferred from one plant to another. .

which changes the pyranose configuration of glucose into the furanose configuration of fructose. a principal chemical is modified by a series of chemical reactions. this step is irreversible. For the metabolic pathway analysis the AraCyc database is used Method Specification: Database: AraCyc (BioCyc) Pathways: 1. The second step is catalyzed by a phosphofructokinase in the presence of ATP. In each pathway. Glycolysis 2. made from starch degradation.Experiment No. The third step is catalyzed by an aldolase which cleaves fructose-1. Glycolysis The pathway starts with β-D-glucose-6-phosphate. The first committed step of glycolysis is the reversible conversion of β-D-glucose-6-phosphate into Dfructose-6-phosphate by hexose phosphate isomerase. Pentose phosphate pathway 3. 3 Aim: To study plant metabolic pathway in Arabidopsis thaliana. Photorespiration Principle Outcome: 1. Principle: Metabolic pathways are series of chemical reactions occurring within a cell.6-bisphosphate into interconvertable .

The following reaction. The next step releases one molecule of ATP during the conversion of 1.3-diphosphateglycerate into 3-phosphoglycerate by a Mg2+-dependent glyceraldehyde-3-phosphate kinase. leading to the release of a molecule of ATP. leading to the formation of 2-phosphoglycerate . is freely reversible and requires NAD+ and phosphate . which adds one phosphate residue to D-glyceraldehyde-3phosphate to form 1.3-diphosphateglycerate . The next step requires little energy change and leads to the reversible transfer of a phosphate group from the 3. . The removal of a molecule of water by an enolase in the presence of Mg2+ converts 2phosphoglycerate into phosphoenolpyruvate (PEP).to the 2-hydroxyl group of glycerate. The final step of glycolysis involves the ketolization of PEP to pyruvate by a pyruvate kinase. The interconversion of these tautomers is facilitated by a triose phosphate isomerase.two three-carbon fragments: D-glyceraldehyde-3-phosphate and dihydroxyacetone phosphate.

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In its dephosphorylated form glycolate is exported to the cytoplasm where it is oxidized in the peroxisomes to glyoxylate . Glyoxylate is then converted into glycine by two different enzymes: serine:glyoxylate aminotransferase and glutamate: glyoxylate aminotransferase. T and L). The methylene group is then transferred to another glycine molecule to form serine by a serine hydroxylmetyltransferase. Back in the peroxisome. The last of the peroxisome steps consists in the reduction of hydroxypyruvate into glycerate by an NADH-dependent hydroxypyruvate reductase. Glycine decarboxylase has four different subunit (P. Glycine is in fact converted into serine in the mitochondrion by glycine decarboxylase where it is extremely abundant. which catalyze the transfer of a methylene group from glycine to tetrahydrofolate with the concomitant release of NH3 and CO2. and production of NADH. Glycerate is then redirected to the chloroplast where it is phosphorylated to 3-phosphoglycerate and reenters the Calvin cycle . serine is used to convert glyoxylate into hydroxypyruvate via serine:glyoxylate aminotransferase as mentioned above. Photorespiration The first step of the pathway involves the dephosphorylation of 2-phosphoglycolate.2. The H2O2 generated during this step is detoxified by catalases in the peroxisome. H.

The subsequent non-oxidative portion represents a series of transaldolase and transketolase reactions. Pentose phosphate pathway The pentose phosphate pathway is an alternative way of oxidizing glucose.3. this step is the source of reducing equivalents for biosynthesis reactions in the shape of NADPH. As a result this pathway is a source of reducing power and is also important for the conversion of hexoses to pentoses Conclusion: We have successfully studied the above three pathways in Arabidopsis thaliana from the AraCyc (BioCyc) pathway database. β-D-glucose-6-phosphate is oxidized to D-ribulose-5phosphate0. This oxidation is coupled with NADPH synthesis. In the former. The pathway has two primary reaction sequences: the pentose phosphate pathway (oxidative branch) and the pentose phosphate pathway (nonoxidative branch). in which D-ribulose-5-phosphate is converted into D-fructose-6phosphate and D-glyceraldehyde-3-phosphate. .

1| Arabidopsis thaliana sph6 gene for S Protein Homologue 6 ATGAATTCGTCTAACATAAATTTCCTAACAATTTTCTACTCAATGTTTATAATCATCTTTATAGTATTAA TATCTTTGATAGGCTGTGAAACTCTACAACATGATGGAAAAGTATTTCCAATGAAAGGTCCTCTTACTAG GGTTGTGATTTATAATGACAATGATTATCTTTTAGGAGTTCATTGTAAATCAAGAGATGATGATCATGGC TTCCATATTCTACAAAAAGGTGGATTATATGGTTGGATGTTTTACGTGAATTTTATGAATTCGACACTCT ACTTCTGTGGATTTAGCCAAGAACAAGTAAAAAAAGGTGTGTTCGATATTTATAAAGCGGTTAGAGATTC TTCTAGATGTAGAAATTGTACTTGGGAAGCAAAGGAAGATGGTATTTATGGATATGGCGAGATTCCTAAG AAAAATCCTTTGTTTTATAAGTGGCTAATGTAA References: 1. Arabidopsis thaliana(Model Plant) >gi|281199944|gb|GU223224. the evolution of the small but essential portion of the genome that actually encodes the organism's genes has proceeded relatively slowly.1| Pisum sativum sulfiredoxin precursor protein (Srx) gene. Hall. BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. taxa that have been reproductively isolated for millions of years have retained recognizable intragenic DNA sequences as well as similar arrangements of genes along the chromosomes. Acids. the study of the similarities and differences in structure and function of hereditary information across taxa. 4 Aim: To comparatively study the plant genomes. T. complete cds ATGGCGGCGAGCAACTTTCTGCTGCAGCTGCCGCTGCGCAGCTTTACCGTGATTAACGTGGCGAGCGCGA GCAGCAGCAACGGTTCGCCGCCGGTGATCGGAGGATCTAGCGGCGGTGTAGGACCGATGATTGTGGAATT ACCGTTGGAGAAGATACGAAGACCGTTGATGCGAACCAGATCCAACGATCAGAACAAAGTGAAAGAGCTT ATGGATAGTATCCGTCAAATCGGTCTTCAAGTTCCGATTGATGTGATTGAAGTTGATGGAACTTACTATG GGTTCTCGGGATGTCACAGATACGAGGCGCATCAGAAGCTAGGGCTTCCAACTATACGTTGCAAAATCCG TAAAGGAACAAAGGAAACATTAAGGCATCATCTTCGCTGA 2. 1999. Ser. uses molecular tools to investigate many notions that long preceded identification of DNA as the hereditary molecule. 41:95-98. double stranded . Symp. Nucl. as a result. Principle: Comparative genomics. Principle Outcomes: DNA molecule: arabidpsis thaliana Length = 453 base pairs Molecular Weight = 136212 Daltons.A. In most plants. single stranded Molecular Weight = 273979 Daltons. Method Specification: Tool: BioEdit Plants: 1. Pisum sativum (Target Plant) >gi|326937563|emb|FN691474.Experiment No.

31 . double stranded G+C content = 50.00% Nucleotide A C G T Number 108 83 112 87 Mol% 27.54 DNA molecule: pisum sativum Length = 390 base pairs Molecular Weight = 118241 Daltons.28 28.65% Nucleotide A C G T Number 150 55 87 161 Mol% 33.00% A+T content = 50.11 12.72 22.21 35. single stranded Molecular Weight = 237117 Daltons.69 21.35% A+T content = 68.G+C content = 31.14 19.

.Pairwise Alignment: Sequence 1: arabidpsis thaliana Sequence 2: pisum sativum Optimal Global aligment Alignment score: 84 Identities: 0.43 Conclusion: We have successfully compared two plant nucleotide sequences from Arabidopsis thaliana and Pisum sativum. we found that the both sequences are 43% identical.

A.5 Aim: To identify the Drug targets in plants.3 Reference: Lupas. the lowest level of information is individual atoms Docking is a method which predicts the preferred orientation of one molecule to a second when bound to each other to form a stable complex. M. Docking is frequently used to predict the binding orientation of small molecule drug candidates to their protein targets in order to in turn predict the affinity and activity of the small molecule. Science 252:1162-1164 . Van Dyke. The common feature of molecular modelling techniques is the atomistic level description of the molecular systems.. (1991) Predicting Coled Coils from Protein Sequences.. and Stock. J.Experiment No. Method Specification: Receptor: 3O74 Ligand: 2XZP Tool: HEX6. Principle: Molecular modelling encompasses all theoretical methods and computational techniques used to model or mimic the behaviour of molecules.

Principle Outcome: Conclusion: We have successfully identified ligand drug target 2XZP for receptor 3O74 of Pseudomonas putida as it shows maximum binding affinity in Docking .

Markowitz S. Wilson A (2011) Geneious v5.6 Aim: To design plant nucleotide primer using primer designing tool. Duran C.5 Reference: Drummond AJ.Experiment No. Sturrock S. Kearse M. and copies the opposite strand. Principle: A primer is a strand of nucleic acid that serves as a starting point for DNA synthesis. Buxton S.1 Tool: Geneious Pro 5. The polymerase starts replication at the 3'-end of the primer. Ashton B. GenBank: U39480.4. DNA polymerases. Cheung M. Available from http://www. Thierer T. They are required for DNA replication because the enzymes that catalyze this process. Cooper A. Stones-Havas S. the primer for DNA synthesis and replication is a short strand of RNA Method Specification: Sequence: Arabidopsis thaliana actin 3 gene.com/ Principle Outcome: . Field M. In most cases of natural DNA replication. Moir R. Heled J. can only add new nucleotides to an existing strand of DNA.3. complete cds.geneious.

complete cds sequence using primer designing tool Geneious Pro 5.3.5 .Type primer_bind primer_bind primer_bind primer_bind primer_bind primer_bind_reverse primer_bind_reverse primer_bind_reverse primer_bind_reverse primer_bind_reverse Name 1st forward primer 2nd forward primer 3rd forward primer 4th forward primer 5th forward primer 1st reverse primer 2nd reverse primer 3rd reverse primer 4th reverse primer 5th reverse primer Sequence TGAGCAGGAGCTTGAGACGGC GCCTTAACCGAGGCCGAGCG GCCTTAACCGAGGCCGAGCG GCCTTAACCGAGGCCGAGCG GCCTTAACCGAGGCCGAGCG ACCTCAGGGCAACGGAAACGC GCTTCGAATTCGGCACACCTCGT GCTTCGAATTCGGCACACCTCG GGCTTCGAATTCGGCACACCT AGGCTTCGAATTCGGCACACC Minimum 2501 848 848 848 848 2591 933 934 936 937 Maximum 2521 867 867 867 867 2611 955 955 956 957 Length 21 20 20 20 20 21 23 22 21 21 # Intervals 1 1 1 1 1 1 1 1 1 1 Direction forward forward forward forward forward reverse reverse reverse reverse reverse Conclusion: We have successfully designed primers for Arabidopsis thaliana actin 3 gene.

1. Principle: Phylogenetic methods can be used for many purposes.7 Aim: To draw phylogram using different plant protein sequence. solanum tuberosum Tool: Geneious 5. Method specification: Plants: 1. oryza sativa 2.7 Principle Outcomes: . including analysis of morphological and several kinds of molecular data. Pseudoterranova decipiens and 3.Experiment No.

'Pseudoterranova decipiens':1. Conclusion: we have successfully drawn phylogram using plant protein sequences of oryza sativa.Tree: + oryza sativa | +========================================================================== ========================= Pseudoterranova decipiens | +=============================== solanum tuberosum In Newick format: ('oryza sativa':0.242189.0.'solanum tuberosum':0.3923650000000001). Pseudoterranova decipiens and solanum tuberosum .

(1991) Predicting Coled Coils from Protein Sequences. maintaining symbiotic associations with soil flora. Secondary metabolites are essentially low molecular weight compounds. communication between plants. A. enhancing the rate of fertilization etc. or reproduction of an organism.. sometimes having complex structures. anti-herbivory.Experiment No. Van Dyke. pollinator attraction. They function in processes as diverse as immunity.. development. Plants use secondary metabolites as medicines. and Stock. M. Secondary metabolites often play an important role in plant defense against herbivory and other interspecies defenses. Principle: Secondary metabolites are organic compounds that are not directly involved in the normal growth.8 Aim: To identify the secondary metabolites as drug targets for diseases in plants. and recreational drugs. J.3 Reference: Lupas. and hence are significant from the evo-devo perspective Method Specification: Receptor: 2XAM (Plant Hydrocarbon) Ligand: 2XAL Tool: HEX6.. Science 252:1162-1164 Principle Outcome: . flavorings.

3. .Conclusion: We have successfully identified ligand drug target 2XZP for receptor 3O74 of Pseudomonas putida as it shows maximum binding affinity in Docking using HEX6.