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
Genome Organization and Evolution:
Arabidopsis thaliana is a diploid plant with 2n = 10 chromosomes. it is an invaluable resource to agriculturally important crops.Arabidopsis thaliana is a small flowering plant of mustard family. which includes canola.924 131 117
Chloroplast Genome Pltd
26-Mar-2010 07-Apr-2000 154. It is now being used as a model organism to study different aspects of plant biology.176
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. It is distributed throughout the world and was first reported in the sixteenth century by Johannes Thal.323 35. Though of no economic importance. It has been used for over fifty years to study plant mutations and for classical genetic analysis.
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. 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.478 129 85
.146. The sequencing was done by an international collaboration collectively termed the Arabidopsis Genome Initiative (AGI). particularly to members of the same family.348 33. brassicaceae (Cruciferae). an important source of vegetable oil. and (3) produces large number of seeds.
140 1 5 . 3.2 0. 14.9 5.507 21 37 131 129
Pltd NC_000932.4 36. 1 2 554
2.1 30.81 36. 14. 3.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
Size other (Mb) GC% protein rrna trna rna gene pseudogene 2 2 3 7 240 218 8.433 96 93 79 149 5.9 8.263 2 NC_003071.2 Y08501.1 23.1 AP000423.35 1 4.107
Nu Chr 1 bottom c arm Nu Chr c 1 top arm
.080 832 948 -
1 NC_003070.710 5 0 AE005173.5 36. 23.043 1. 3.228 31 92 8 4 BA000015.140 924 1.089 MT NC_001284.323 1 1 AJ270060.1 26.730 116 5. 14.46 36.15 36. 3.43 35.40 6 1.8 117 85
123 127 7.3
Arabidopsis Genome Initiative Arabidopsis thaliana legacy genome sequence
Typ RefSe e Name q 3 -
Size GC protei rrn trn other pseudogen (Mb) % n a a rna gene e 6 4.1 19.59 36.128
AE005172.8 CP002688.7 CP002685.3 6. 4.72 7 817
Nu Chr 4 long c arm Nu Chr 4 short c arm
.22 36.356 5 NC_003076.2 5.7 35.71 1 79
Nu Chr c Nu Chr c
BA000014.156 1 0 AJ270058.9 CP002684.98 35.37 44.513 134 6.83 0 2.67 35.1 0. 23.05 36.560 3 NC_003074.8 CP002686.908 4 NC_003075.7 CP002687.9 9. 5.1 18.
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.
. Dutta S.Experiment No. Singhal P.. J. 46(1). and Jayaram B. 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. Chem. Agrawal P. Kritee.. A Physico-Chemical model for analyzing DNA sequences".0”software. Inf.0 Reference:
1. Khurana E. 78-85.. Jayaram B. Read more about ChemGenome Procedure:
Genome: Arabidopsis thaliana chromosome1 Tool: ChemGenome 2. The methodology follows a physico-chemical approach and has been validated on 372 prokaryotic genomes. 2. Tomer R. Chemgenome is an ab-intio gene prediction software. 2008. Mod... "Decoding the design principles of amino acids and the chemical logic of protein sequences"... Nature Precedings.
Conclusion: We have successfully identifed gene in Arabidopsis thaliana which can be transferred from one plant to another.
Glycolysis 2. The first committed step of glycolysis is the reversible conversion of β-D-glucose-6-phosphate into Dfructose-6-phosphate by hexose phosphate isomerase.6-bisphosphate into interconvertable
. Photorespiration Principle Outcome: 1. Principle: Metabolic pathways are series of chemical reactions occurring within a cell. this step is irreversible. a principal chemical is modified by a series of chemical reactions. made from starch degradation. The second step is catalyzed by a phosphofructokinase in the presence of ATP. which changes the pyranose configuration of glucose into the furanose configuration of fructose. The third step is catalyzed by an aldolase which cleaves fructose-1. Pentose phosphate pathway 3. For the metabolic pathway analysis the AraCyc database is used Method Specification: Database: AraCyc (BioCyc)
Pathways: 1. Glycolysis The pathway starts with β-D-glucose-6-phosphate. In each pathway. 3 Aim: To study plant metabolic pathway in Arabidopsis thaliana.Experiment No.
two three-carbon fragments: D-glyceraldehyde-3-phosphate and dihydroxyacetone phosphate. The final step of glycolysis involves the ketolization of PEP to pyruvate by a pyruvate kinase.to the 2-hydroxyl group of glycerate.3-diphosphateglycerate into 3-phosphoglycerate by a Mg2+-dependent glyceraldehyde-3-phosphate kinase. leading to the formation of 2-phosphoglycerate .3-diphosphateglycerate . leading to the release of a molecule of ATP. The interconversion of these tautomers is facilitated by a triose phosphate isomerase. is freely reversible and requires NAD+ and phosphate . The next step releases one molecule of ATP during the conversion of 1. The removal of a molecule of water by an enolase in the presence of Mg2+ converts 2phosphoglycerate into phosphoenolpyruvate (PEP). The following reaction. The next step requires little energy change and leads to the reversible transfer of a phosphate group from the 3. which adds one phosphate residue to D-glyceraldehyde-3phosphate to form 1.
Glycerate is then redirected to the chloroplast where it is phosphorylated to 3-phosphoglycerate and reenters the Calvin cycle
. In its dephosphorylated form glycolate is exported to the cytoplasm where it is oxidized in the peroxisomes to glyoxylate . and production of NADH. The last of the peroxisome steps consists in the reduction of hydroxypyruvate into glycerate by an NADH-dependent hydroxypyruvate reductase. The methylene group is then transferred to another glycine molecule to form serine by a serine hydroxylmetyltransferase. which catalyze the transfer of a methylene group from glycine to tetrahydrofolate with the concomitant release of NH3 and CO2. serine is used to convert glyoxylate into hydroxypyruvate via serine:glyoxylate aminotransferase as mentioned above. Back in the peroxisome. Glycine is in fact converted into serine in the mitochondrion by glycine decarboxylase where it is extremely abundant. The H2O2 generated during this step is detoxified by catalases in the peroxisome. Glyoxylate is then converted into glycine by two different enzymes: serine:glyoxylate aminotransferase and glutamate: glyoxylate aminotransferase. H.2. Photorespiration The first step of the pathway involves the dephosphorylation of 2-phosphoglycolate. Glycine decarboxylase has four different subunit (P. T and L).
β-D-glucose-6-phosphate is oxidized to D-ribulose-5phosphate0.3. in which D-ribulose-5-phosphate is converted into D-fructose-6phosphate and D-glyceraldehyde-3-phosphate. This oxidation is coupled with NADPH synthesis. Pentose phosphate pathway The pentose phosphate pathway is an alternative way of oxidizing glucose. this step is the source of reducing equivalents for biosynthesis reactions in the shape of NADPH. In the former.
. 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. The pathway has two primary reaction sequences: the pentose phosphate pathway (oxidative branch) and the pentose phosphate pathway (nonoxidative branch). The subsequent non-oxidative portion represents a series of transaldolase and transketolase reactions.
41:95-98. as a result. Arabidopsis thaliana(Model Plant)
>gi|281199944|gb|GU223224. BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Acids. Symp. Principle: Comparative genomics. 1999. Ser. Hall. double stranded
.1| Arabidopsis thaliana sph6 gene for S Protein Homologue 6 ATGAATTCGTCTAACATAAATTTCCTAACAATTTTCTACTCAATGTTTATAATCATCTTTATAGTATTAA TATCTTTGATAGGCTGTGAAACTCTACAACATGATGGAAAAGTATTTCCAATGAAAGGTCCTCTTACTAG GGTTGTGATTTATAATGACAATGATTATCTTTTAGGAGTTCATTGTAAATCAAGAGATGATGATCATGGC TTCCATATTCTACAAAAAGGTGGATTATATGGTTGGATGTTTTACGTGAATTTTATGAATTCGACACTCT ACTTCTGTGGATTTAGCCAAGAACAAGTAAAAAAAGGTGTGTTCGATATTTATAAAGCGGTTAGAGATTC TTCTAGATGTAGAAATTGTACTTGGGAAGCAAAGGAAGATGGTATTTATGGATATGGCGAGATTCCTAAG AAAAATCCTTTGTTTTATAAGTGGCTAATGTAA
References: 1. Pisum sativum (Target Plant)
>gi|326937563|emb|FN691474. Method Specification: Tool: BioEdit Plants: 1. single stranded Molecular Weight = 273979 Daltons.
DNA molecule: arabidpsis thaliana Length = 453 base pairs Molecular Weight = 136212 Daltons. the evolution of the small but essential portion of the genome that actually encodes the organism's genes has proceeded relatively slowly. uses molecular tools to investigate many notions that long preceded identification of DNA as the hereditary molecule. Nucl. 4 Aim: To comparatively study the plant genomes. T.Experiment No.A.1| Pisum sativum sulfiredoxin precursor protein (Srx) gene. complete cds ATGGCGGCGAGCAACTTTCTGCTGCAGCTGCCGCTGCGCAGCTTTACCGTGATTAACGTGGCGAGCGCGA GCAGCAGCAACGGTTCGCCGCCGGTGATCGGAGGATCTAGCGGCGGTGTAGGACCGATGATTGTGGAATT ACCGTTGGAGAAGATACGAAGACCGTTGATGCGAACCAGATCCAACGATCAGAACAAAGTGAAAGAGCTT ATGGATAGTATCCGTCAAATCGGTCTTCAAGTTCCGATTGATGTGATTGAAGTTGATGGAACTTACTATG GGTTCTCGGGATGTCACAGATACGAGGCGCATCAGAAGCTAGGGCTTCCAACTATACGTTGCAAAATCCG TAAAGGAACAAAGGAAACATTAAGGCATCATCTTCGCTGA
2. In most plants. 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. the study of the similarities and differences in structure and function of hereditary information across taxa.
11 12.21 35. single stranded Molecular Weight = 237117 Daltons.14 19. double stranded G+C content = 50.65% Nucleotide A C G T Number 150 55 87 161 Mol% 33.00% 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.00% A+T content = 50.G+C content = 31.28 28.31
.35% A+T content = 68.69 21.72 22.
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.
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. A. J.Experiment No. Science 252:1162-1164
. 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. (1991) Predicting Coled Coils from Protein Sequences. 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..5 Aim: To identify the Drug targets in plants. and Stock. M. The common feature of molecular modelling techniques is the atomistic level description of the molecular systems.3 Reference: Lupas. Van Dyke.
Conclusion: We have successfully identified ligand drug target 2XZP for receptor 3O74 of Pseudomonas putida as it shows maximum binding affinity in Docking
Cheung M. Thierer T. The polymerase starts replication at the 3'-end of the primer.5 Reference: Drummond AJ. Kearse M. Principle: A primer is a strand of nucleic acid that serves as a starting point for DNA synthesis. Ashton B. Heled J. Buxton S. and copies the opposite strand. Moir R.geneious.4. They are required for DNA replication because the enzymes that catalyze this process. Field M. In most cases of natural DNA replication.6 Aim: To design plant nucleotide primer using primer designing tool.3. Sturrock S. can only add new nucleotides to an existing strand of DNA. Duran C.Experiment No. Cooper A.1 Tool: Geneious Pro 5. complete cds. Stones-Havas S. Wilson A (2011) Geneious v5. GenBank: U39480.com/ Principle Outcome:
. Available from http://www. Markowitz S. DNA polymerases. the primer for DNA synthesis and replication is a short strand of RNA Method Specification: Sequence: Arabidopsis thaliana actin 3 gene.
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.3. complete cds sequence using primer designing tool Geneious Pro 5.5
7 Principle Outcomes:
. solanum tuberosum Tool: Geneious 5.1.7 Aim: To draw phylogram using different plant protein sequence. oryza sativa 2. Method specification: Plants: 1. Principle: Phylogenetic methods can be used for many purposes. including analysis of morphological and several kinds of molecular data.Experiment No. Pseudoterranova decipiens and 3.
Conclusion: we have successfully drawn phylogram using plant protein sequences of oryza sativa. Pseudoterranova decipiens and solanum tuberosum
+ oryza sativa | +========================================================================== ========================= Pseudoterranova decipiens | +=============================== solanum tuberosum
In Newick format:
sometimes having complex structures. Science 252:1162-1164 Principle Outcome:
. Principle: Secondary metabolites are organic compounds that are not directly involved in the normal growth. A. Secondary metabolites often play an important role in plant defense against herbivory and other interspecies defenses. communication between plants. (1991) Predicting Coled Coils from Protein Sequences. They function in processes as diverse as immunity. J. Secondary metabolites are essentially low molecular weight compounds. and hence are significant from the evo-devo perspective Method Specification: Receptor: 2XAM (Plant Hydrocarbon) Ligand: 2XAL Tool: HEX6.Experiment No. flavorings.3 Reference: Lupas. and Stock..8 Aim: To identify the secondary metabolites as drug targets for diseases in plants.. and recreational drugs. maintaining symbiotic associations with soil flora. development. anti-herbivory. enhancing the rate of fertilization etc. pollinator attraction.. Van Dyke. or reproduction of an organism. M. Plants use secondary metabolites as medicines.
.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.