Msc tics Practical.

8/2/2019 Msc tics Practical.

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PLANT GENOMICS


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Experiment No.1

Aim: To study the plant genome organization and evolution ofArabidopsis thaliana.

Principle:

The plant genome organization describes the genomic size, chromosome number, repeatsequences, 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-dp-sis tha-li--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 changedthis 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 NCBIresources(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
http://en.wikipedia.org/wiki/Molecular_biologyhttp://en.wikipedia.org/wiki/Flowerhttp://en.wikipedia.org/wiki/Phototropismhttp://en.wikipedia.org/wiki/Syllable_stress_of_Botanical_Latinhttp://en.wikipedia.org/wiki/Syllable_stress_of_Botanical_Latinhttp://en.wikipedia.org/wiki/Syllable_stress_of_Botanical_Latinhttp://en.wikipedia.org/wiki/Flowering_planthttp://www.ncbi.nlm.nih.gov/genome/http://www.ncbi.nlm.nih.gov/genome/http://www.ncbi.nlm.nih.gov/genome/http://www.ncbi.nlm.nih.gov/genome/http://en.wikipedia.org/wiki/Flowering_planthttp://en.wikipedia.org/wiki/Syllable_stress_of_Botanical_Latinhttp://en.wikipedia.org/wiki/Phototropismhttp://en.wikipedia.org/wiki/Flowerhttp://en.wikipedia.org/wiki/Molecular_biology


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Plant Resource:

Genome Organization and Evolution:


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Arabidopsis thaliana is a small flowering plant of mustard family, brassicaceae (Cruciferae).

It is distributed throughout the world and was first reported in the sixteenth century by

Johannes Thal. It has been used for over fifty years to study plant mutations and for classical

genetic analysis. It is now being used as a model organism to study different aspects of plant

biology.

Arabidopsis thaliana is a diploid plant with 2n = 10 chromosomes. It became the first plantgenome 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, and (3) produces large number of seeds. The sequencing

was done by an international collaboration collectively termed the Arabidopsis Genome

Initiative (AGI). Though of no economic importance, it is an invaluable resource to

agriculturally important crops, particularly to members of the same family, which includes

canola, an important source of vegetable oil.

Genome Assembly and Annotation:

Assembly and Annotation

Default assembly

1 other assemblies are availableAssembly Name TAIR9

Last sequence update 19-Jun-2009

Highest level of assembly complete sequence genome

Size (total bases) 119,146,348

Number of genes 33,323

Number of proteins 35,176

Mitochondrial Genome

Last record update 31-Jul-2008Last sequence update 12-Dec-2002Size 366,924Number of genes 131

Number of proteins 117Chloroplast Genome Pltd

Last record update 26-Mar-2010Last sequence update 07-Apr-2000Size 154,478Number of genes 129Number of proteins 85

Arabidopsis thaliana
http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?id=3702http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?id=3702http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?id=3702


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The Arabidopsis Information Resource (TAIR)Arabidopsis assembly project

Loc Type

Na

me RefSeq INSDC

Size

(Mb) GC% protein rrna trna

other

rna gene pseudogene

Nuc Chr 1 NC_003070.9 CP002684.1 30.43 35.9 9,263 - 240 218 8,433 924

Nuc Chr 2 NC_003071.7 CP002685.1 19.7 35.9 5,560 2 96 149 5,513 1,043

Nuc Chr 3 NC_003074.8 CP002686.1 23.46 36.3 6,908 2 93 134 6,730 1,080

Nuc Chr 4 NC_003075.7 CP002687.1 18.59 36.2 5,356 - 79 116 5,140 832

Nuc Chr 5 NC_003076.8 CP002688.1 26.98 35.9 8,089 - 123 127 7,507 948

MT Chr MT NC_001284.2 Y08501.2 0.37 44.8 117 3 21 - 131 -

Chl Chr Pltd NC_000932.1 AP000423.1 0.15 36.3 85 7 37 - 129 -

Arabidopsis thaliana

Arabidopsis Genome InitiativeArabidopsis thaliana legacy genome sequence

Loc

Typ

e Name

RefSe

q INSDC

Size

(Mb)

GC

%

protei

n

rrn

a

trn

a

other

rna gene

pseudogen

e

Nu

c

Chr 3 - BA000014.

8

23.4 36.

4

5,228 31 92 6 4,40

6

79

Nu

c

Chr 5 - BA000015.

5

23.81 36.

0

4,710 - 107 10 1,83

0

-

Nu

c

Chr 1

bottom

arm

- AE005173.

1

14.67 35.

5

3,140 - 128 - 2,71

1

7

Nu

c

Chr 1 top

arm

- AE005172.

1

14.22 36.

1

3,323 1 78 1 2,35

1

5

Nu

c

Chr 4 long

arm

- AJ270060.

1

14.5 36.

0

3,156 - 55 - 4,72

7

-

Nu

c

Chr 4 short

arm

- AJ270058.

1

3.05 36.

2

554 - 8 - 817 -
http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?id=3702http://www.arabidopsis.org/http://www.arabidopsis.org/http://www.ncbi.nlm.nih.gov/nuccore/240254421http://www.ncbi.nlm.nih.gov/nuccore/240254421http://www.ncbi.nlm.nih.gov/nuccore/332189094http://www.ncbi.nlm.nih.gov/nuccore/332189094http://www.ncbi.nlm.nih.gov/nuccore/240254678http://www.ncbi.nlm.nih.gov/nuccore/240254678http://www.ncbi.nlm.nih.gov/nuccore/330250293http://www.ncbi.nlm.nih.gov/nuccore/330250293http://www.ncbi.nlm.nih.gov/nuccore/240255695http://www.ncbi.nlm.nih.gov/nuccore/240255695http://www.ncbi.nlm.nih.gov/nuccore/332640072http://www.ncbi.nlm.nih.gov/nuccore/332640072http://www.ncbi.nlm.nih.gov/nuccore/240256243http://www.ncbi.nlm.nih.gov/nuccore/240256243http://www.ncbi.nlm.nih.gov/nuccore/332656411http://www.ncbi.nlm.nih.gov/nuccore/332656411http://www.ncbi.nlm.nih.gov/nuccore/240256493http://www.ncbi.nlm.nih.gov/nuccore/240256493http://www.ncbi.nlm.nih.gov/nuccore/332002898http://www.ncbi.nlm.nih.gov/nuccore/332002898http://www.ncbi.nlm.nih.gov/nuccore/26556996http://www.ncbi.nlm.nih.gov/nuccore/26556996http://www.ncbi.nlm.nih.gov/nuccore/49256807http://www.ncbi.nlm.nih.gov/nuccore/49256807http://www.ncbi.nlm.nih.gov/nuccore/7525012http://www.ncbi.nlm.nih.gov/nuccore/7525012http://www.ncbi.nlm.nih.gov/nuccore/7525012http://www.ncbi.nlm.nih.gov/nuccore/5881673http://www.ncbi.nlm.nih.gov/nuccore/5881673http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?id=3702http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?id=3702http://www.arabidopsis.org/info/agi.jsphttp://www.arabidopsis.org/info/agi.jsphttp://www.ncbi.nlm.nih.gov/nuccore/55417891http://www.ncbi.nlm.nih.gov/nuccore/55417891http://www.ncbi.nlm.nih.gov/nuccore/55417889http://www.ncbi.nlm.nih.gov/nuccore/55417889http://www.ncbi.nlm.nih.gov/nuccore/12063652http://www.ncbi.nlm.nih.gov/nuccore/12063652http://www.ncbi.nlm.nih.gov/nuccore/12063420http://www.ncbi.nlm.nih.gov/nuccore/12063420http://www.ncbi.nlm.nih.gov/nuccore/42494966http://www.ncbi.nlm.nih.gov/nuccore/42494966http://www.ncbi.nlm.nih.gov/nuccore/42494965http://www.ncbi.nlm.nih.gov/nuccore/42494965http://www.ncbi.nlm.nih.gov/nuccore/42494965http://www.ncbi.nlm.nih.gov/nuccore/42494965http://www.ncbi.nlm.nih.gov/nuccore/42494966http://www.ncbi.nlm.nih.gov/nuccore/42494966http://www.ncbi.nlm.nih.gov/nuccore/12063420http://www.ncbi.nlm.nih.gov/nuccore/12063420http://www.ncbi.nlm.nih.gov/nuccore/12063652http://www.ncbi.nlm.nih.gov/nuccore/12063652http://www.ncbi.nlm.nih.gov/nuccore/55417889http://www.ncbi.nlm.nih.gov/nuccore/55417889http://www.ncbi.nlm.nih.gov/nuccore/55417891http://www.ncbi.nlm.nih.gov/nuccore/55417891http://www.arabidopsis.org/info/agi.jsphttp://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?id=3702http://www.ncbi.nlm.nih.gov/nuccore/5881673http://www.ncbi.nlm.nih.gov/nuccore/7525012http://www.ncbi.nlm.nih.gov/nuccore/49256807http://www.ncbi.nlm.nih.gov/nuccore/26556996http://www.ncbi.nlm.nih.gov/nuccore/332002898http://www.ncbi.nlm.nih.gov/nuccore/240256493http://www.ncbi.nlm.nih.gov/nuccore/332656411http://www.ncbi.nlm.nih.gov/nuccore/240256243http://www.ncbi.nlm.nih.gov/nuccore/332640072http://www.ncbi.nlm.nih.gov/nuccore/240255695http://www.ncbi.nlm.nih.gov/nuccore/330250293http://www.ncbi.nlm.nih.gov/nuccore/240254678http://www.ncbi.nlm.nih.gov/nuccore/332189094http://www.ncbi.nlm.nih.gov/nuccore/240254421http://www.arabidopsis.org/


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Conclusion:

The genome organization and evolution ofArabiopsis thaliana was successfully studied. I

found thatArabidopsis thaliana is a small flowering plant of mustard family having 10chromosomes with ~ 120 Mb genome size.


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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

ChemGenome2.0software.

Chemgenome is an ab-intio gene prediction software, which find genes in prokaryotic

genomes in all six reading frames. The methodology follows a physico-chemical approach

and has been validated on 372 prokaryotic genomes. Read more about ChemGenome

Procedure:

Genome: Arabidopsis thaliana chromosome1

Tool: ChemGenome 2.0

Reference:

1. A Physico-Chemical model for analyzing DNA sequences", Dutta S., Singhal P.,Agrawal P., Tomer R., Kritee, Khurana E. and Jayaram B., J. Chem. Inf. Mod., 2006,

46(1), 78-85.

2. "Decoding the design principles of amino acids and the chemical logic of proteinsequences", Jayaram B., Nature Precedings, 2008.
http://www.scfbio-iitd.res.in/research/genepredictor.htmhttp://www.scfbio-iitd.res.in/research/genepredictor.htmhttp://www.scfbio-iitd.res.in/research/genepredictor.htm


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Principle Outcome:

Conclusion:

We have successfully identifed gene in Arabidopsis thaliana which can be transferred from

one plant to another.


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Experiment No. 3

Aim: To study plant metabolic pathway inArabidopsis thaliana.

Principle:

Metabolic pathways are series ofchemical reactions occurring within a cell. In each pathway,a principal chemical is modified by a series ofchemical reactions. For the metabolic pathway

analysis the AraCyc database is used

Method Specification:

Database: AraCyc (BioCyc)

Pathways:

1. Glycolysis

2. Pentose phosphate pathway3. Photorespiration

Principle Outcome:

1. Glycolysis

The pathway starts with-D-glucose-6-phosphate, made from starch degradation. The first

committed step of glycolysis is the reversible conversion of-D-glucose-6-phosphate into D-

fructose-6-phosphate by hexose phosphate isomerase, which changes the pyranose

configuration of glucose into the furanose configuration of fructose. The second step is

catalyzed by a phosphofructokinase in the presence ofATP; this step is irreversible. The third

step is catalyzed by an aldolase which cleaves fructose-1,6-bisphosphate into interconvertable
http://en.wikipedia.org/wiki/Chemistryhttp://en.wikipedia.org/wiki/Cell_(biology)http://en.wikipedia.org/wiki/Chemical_reactionhttp://biocyc.org/ARA/NEW-IMAGE?type=COMPOUND&object=GLC-6-Phttp://biocyc.org/ARA/NEW-IMAGE?type=COMPOUND&object=GLC-6-Phttp://biocyc.org/ARA/NEW-IMAGE?type=COMPOUND&object=GLC-6-Phttp://biocyc.org/ARA/NEW-IMAGE?type=PATHWAY&object=PWY-842http://biocyc.org/ARA/NEW-IMAGE?type=COMPOUND&object=GLC-6-Phttp://biocyc.org/ARA/NEW-IMAGE?type=COMPOUND&object=GLC-6-Phttp://biocyc.org/ARA/NEW-IMAGE?type=COMPOUND&object=GLC-6-Phttp://biocyc.org/ARA/NEW-IMAGE?type=COMPOUND&object=FRUCTOSE-6Phttp://biocyc.org/ARA/NEW-IMAGE?type=COMPOUND&object=FRUCTOSE-6Phttp://biocyc.org/ARA/NEW-IMAGE?type=COMPOUND&object=ATPhttp://biocyc.org/ARA/NEW-IMAGE?type=COMPOUND&object=FRUCTOSE-16-DIPHOSPHATEhttp://biocyc.org/ARA/NEW-IMAGE?type=COMPOUND&object=FRUCTOSE-16-DIPHOSPHATEhttp://biocyc.org/ARA/NEW-IMAGE?type=COMPOUND&object=ATPhttp://biocyc.org/ARA/NEW-IMAGE?type=COMPOUND&object=FRUCTOSE-6Phttp://biocyc.org/ARA/NEW-IMAGE?type=COMPOUND&object=FRUCTOSE-6Phttp://biocyc.org/ARA/NEW-IMAGE?type=COMPOUND&object=GLC-6-Phttp://biocyc.org/ARA/NEW-IMAGE?type=PATHWAY&object=PWY-842http://biocyc.org/ARA/NEW-IMAGE?type=COMPOUND&object=GLC-6-Phttp://en.wikipedia.org/wiki/Chemical_reactionhttp://en.wikipedia.org/wiki/Cell_(biology)http://en.wikipedia.org/wiki/Chemistry


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two three-carbon fragments: D-glyceraldehyde-3-phosphate and dihydroxyacetone

phosphate. The interconversion of these tautomers is facilitated by a triose phosphate

isomerase. The following reaction, which adds one phosphate residue to D-glyceraldehyde-3-

phosphate to form 1,3-diphosphateglycerate , is freely reversible and

requires NAD+

and phosphate . The next step releases one molecule of ATP during the

conversion of1,3-diphosphateglycerate into 3-phosphoglycerate by a Mg2+

-dependentglyceraldehyde-3-phosphate kinase. The next step requires little energy change and leads to

the reversible transfer of a phosphate group from the 3- to the 2-hydroxyl group of glycerate,

leading to the formation of2-phosphoglycerate . The removal of a molecule of water by an

enolase in the presence of Mg2+

converts 2-

phosphoglycerate into phosphoenolpyruvate (PEP). The final step of glycolysis involves the

ketolization of PEP to pyruvate by a pyruvate kinase, leading to the release of a molecule of

ATP.
http://biocyc.org/ARA/NEW-IMAGE?type=COMPOUND&object=GAPhttp://biocyc.org/ARA/NEW-IMAGE?type=COMPOUND&object=DIHYDROXY-ACETONE-PHOSPHATEhttp://biocyc.org/ARA/NEW-IMAGE?type=COMPOUND&object=DIHYDROXY-ACETONE-PHOSPHATEhttp://biocyc.org/ARA/NEW-IMAGE?type=COMPOUND&object=GAPhttp://biocyc.org/ARA/NEW-IMAGE?type=COMPOUND&object=GAPhttp://biocyc.org/ARA/NEW-IMAGE?type=COMPOUND&object=DPGhttp://biocyc.org/ARA/NEW-IMAGE?type=COMPOUND&object=NADhttp://biocyc.org/ARA/NEW-IMAGE?type=COMPOUND&object=NADhttp://biocyc.org/ARA/NEW-IMAGE?type=COMPOUND&object=NADhttp://biocyc.org/ARA/NEW-IMAGE?type=COMPOUND&object=Pihttp://biocyc.org/ARA/NEW-IMAGE?type=COMPOUND&object=DPGhttp://biocyc.org/ARA/NEW-IMAGE?type=COMPOUND&object=G3Phttp://biocyc.org/ARA/NEW-IMAGE?type=COMPOUND&object=2-PGhttp://biocyc.org/ARA/NEW-IMAGE?type=COMPOUND&object=2-PGhttp://biocyc.org/ARA/NEW-IMAGE?type=COMPOUND&object=2-PGhttp://biocyc.org/ARA/NEW-IMAGE?type=COMPOUND&object=PHOSPHO-ENOL-PYRUVATEhttp://biocyc.org/ARA/NEW-IMAGE?type=COMPOUND&object=PHOSPHO-ENOL-PYRUVATEhttp://biocyc.org/ARA/NEW-IMAGE?type=COMPOUND&object=PHOSPHO-ENOL-PYRUVATEhttp://biocyc.org/ARA/NEW-IMAGE?type=COMPOUND&object=PYRUVATEhttp://biocyc.org/ARA/NEW-IMAGE?type=COMPOUND&object=PYRUVATEhttp://biocyc.org/ARA/NEW-IMAGE?type=COMPOUND&object=PHOSPHO-ENOL-PYRUVATEhttp://biocyc.org/ARA/NEW-IMAGE?type=COMPOUND&object=2-PGhttp://biocyc.org/ARA/NEW-IMAGE?type=COMPOUND&object=2-PGhttp://biocyc.org/ARA/NEW-IMAGE?type=COMPOUND&object=2-PGhttp://biocyc.org/ARA/NEW-IMAGE?type=COMPOUND&object=G3Phttp://biocyc.org/ARA/NEW-IMAGE?type=COMPOUND&object=DPGhttp://biocyc.org/ARA/NEW-IMAGE?type=COMPOUND&object=Pihttp://biocyc.org/ARA/NEW-IMAGE?type=COMPOUND&object=NADhttp://biocyc.org/ARA/NEW-IMAGE?type=COMPOUND&object=DPGhttp://biocyc.org/ARA/NEW-IMAGE?type=COMPOUND&object=GAPhttp://biocyc.org/ARA/NEW-IMAGE?type=COMPOUND&object=GAPhttp://biocyc.org/ARA/NEW-IMAGE?type=COMPOUND&object=DIHYDROXY-ACETONE-PHOSPHATEhttp://biocyc.org/ARA/NEW-IMAGE?type=COMPOUND&object=DIHYDROXY-ACETONE-PHOSPHATEhttp://biocyc.org/ARA/NEW-IMAGE?type=COMPOUND&object=GAP


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2. PhotorespirationThe first step of the pathway involves the dephosphorylation of2-phosphoglycolate, In its

dephosphorylated form glycolate is exported to the cytoplasm where it is oxidized in the

peroxisomes to glyoxylate . 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. Glycine is infact converted into serine in the mitochondrion by glycine decarboxylase where it is

extremely abundant. Glycine decarboxylase has four different subunit (P, H, T and L), which

catalyze the transfer of a methylene group from glycine to tetrahydrofolate with the

concomitant release of NH3 and CO2, and production of NADH. The methylene group is then

transferred to another glycine molecule to form serine by a serine hydroxylmetyltransferase.

Back in the peroxisome, serine is used to convert glyoxylate into hydroxypyruvate via

serine:glyoxylate aminotransferase as mentioned above. The last of the peroxisome steps

consists in the reduction of hydroxypyruvate into glycerate by an NADH-dependent

hydroxypyruvate reductase. Glycerate is then redirected to the chloroplast where it is

phosphorylated to 3-phosphoglycerate and reenters the Calvin cycle
http://biocyc.org/ARA/NEW-IMAGE?type=COMPOUND&object=CPD-67http://biocyc.org/ARA/NEW-IMAGE?type=COMPOUND&object=GLYCOLLATEhttp://biocyc.org/ARA/NEW-IMAGE?type=COMPOUND&object=GLYOXhttp://biocyc.org/ARA/NEW-IMAGE?type=COMPOUND&object=HYDROGEN-PEROXIDEhttp://biocyc.org/ARA/NEW-IMAGE?type=COMPOUND&object=HYDROGEN-PEROXIDEhttp://biocyc.org/ARA/NEW-IMAGE?type=COMPOUND&object=HYDROGEN-PEROXIDEhttp://biocyc.org/ARA/NEW-IMAGE?type=COMPOUND&object=HYDROGEN-PEROXIDEhttp://biocyc.org/ARA/NEW-IMAGE?type=COMPOUND&object=HYDROGEN-PEROXIDEhttp://biocyc.org/ARA/NEW-IMAGE?type=COMPOUND&object=GLYhttp://biocyc.org/ARA/NEW-IMAGE?type=COMPOUND&object=THFhttp://biocyc.org/ARA/NEW-IMAGE?type=COMPOUND&object=OH-PYRhttp://biocyc.org/ARA/NEW-IMAGE?type=COMPOUND&object=GLYCERATEhttp://biocyc.org/ARA/NEW-IMAGE?type=COMPOUND&object=G3Phttp://biocyc.org/ARA/NEW-IMAGE?type=COMPOUND&object=G3Phttp://biocyc.org/ARA/NEW-IMAGE?type=COMPOUND&object=GLYCERATEhttp://biocyc.org/ARA/NEW-IMAGE?type=COMPOUND&object=OH-PYRhttp://biocyc.org/ARA/NEW-IMAGE?type=COMPOUND&object=THFhttp://biocyc.org/ARA/NEW-IMAGE?type=COMPOUND&object=GLYhttp://biocyc.org/ARA/NEW-IMAGE?type=COMPOUND&object=HYDROGEN-PEROXIDEhttp://biocyc.org/ARA/NEW-IMAGE?type=COMPOUND&object=GLYOXhttp://biocyc.org/ARA/NEW-IMAGE?type=COMPOUND&object=GLYCOLLATEhttp://biocyc.org/ARA/NEW-IMAGE?type=COMPOUND&object=CPD-67


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3. Pentose phosphate pathwayThe pentose phosphate pathway is an alternative way of oxidizing glucose. This oxidation is

coupled with NADPH synthesis. The pathway has two primary reaction sequences:

the pentose phosphate pathway (oxidative branch) and the pentose phosphate pathway (non-

oxidative branch). In the former,-D-glucose-6-phosphate is oxidized to D-ribulose-5-

phosphate0; this step is the source of reducing equivalents for biosynthesis reactions in theshape ofNADPH. The subsequent non-oxidative portion represents a series of transaldolase

and transketolase reactions, in which D-ribulose-5-phosphate is converted into D-fructose-6-

phosphate and D-glyceraldehyde-3-phosphate. 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 theAraCyc (BioCyc) pathway database.
http://biocyc.org/ARA/NEW-IMAGE?type=PATHWAY&object=OXIDATIVEPENT-PWYhttp://biocyc.org/ARA/NEW-IMAGE?type=PATHWAY&object=NONOXIPENT-PWYhttp://biocyc.org/ARA/NEW-IMAGE?type=PATHWAY&object=NONOXIPENT-PWYhttp://biocyc.org/ARA/NEW-IMAGE?type=COMPOUND&object=GLC-6-Phttp://biocyc.org/ARA/NEW-IMAGE?type=COMPOUND&object=GLC-6-Phttp://biocyc.org/ARA/NEW-IMAGE?type=COMPOUND&object=GLC-6-Phttp://biocyc.org/ARA/NEW-IMAGE?type=COMPOUND&object=RIBULOSE-5Phttp://biocyc.org/ARA/NEW-IMAGE?type=COMPOUND&object=RIBULOSE-5Phttp://biocyc.org/ARA/NEW-IMAGE?type=COMPOUND&object=NADPHhttp://biocyc.org/ARA/NEW-IMAGE?type=COMPOUND&object=RIBULOSE-5Phttp://biocyc.org/ARA/NEW-IMAGE?type=COMPOUND&object=FRUCTOSE-6Phttp://biocyc.org/ARA/NEW-IMAGE?type=COMPOUND&object=FRUCTOSE-6Phttp://biocyc.org/ARA/NEW-IMAGE?type=COMPOUND&object=GAPhttp://biocyc.org/ARA/NEW-IMAGE?type=COMPOUND&object=GAPhttp://biocyc.org/ARA/NEW-IMAGE?type=COMPOUND&object=FRUCTOSE-6Phttp://biocyc.org/ARA/NEW-IMAGE?type=COMPOUND&object=FRUCTOSE-6Phttp://biocyc.org/ARA/NEW-IMAGE?type=COMPOUND&object=RIBULOSE-5Phttp://biocyc.org/ARA/NEW-IMAGE?type=COMPOUND&object=NADPHhttp://biocyc.org/ARA/NEW-IMAGE?type=COMPOUND&object=RIBULOSE-5Phttp://biocyc.org/ARA/NEW-IMAGE?type=COMPOUND&object=RIBULOSE-5Phttp://biocyc.org/ARA/NEW-IMAGE?type=COMPOUND&object=GLC-6-Phttp://biocyc.org/ARA/NEW-IMAGE?type=PATHWAY&object=NONOXIPENT-PWYhttp://biocyc.org/ARA/NEW-IMAGE?type=PATHWAY&object=NONOXIPENT-PWYhttp://biocyc.org/ARA/NEW-IMAGE?type=PATHWAY&object=OXIDATIVEPENT-PWY


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Experiment No. 4

Aim: To comparatively study the plant genomes.

Principle:Comparative genomics, the study of the similarities and differences in structureand function of hereditary information across taxa, uses molecular tools to investigate manynotions that long preceded identification of DNA as the hereditary molecule. In most plants,

the evolution of the small but essential portion of the genome that actually encodes the

organism's genes has proceeded relatively slowly; as a result, 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.


Tool: BioEdit

Plants:1. Arabidopsis thaliana(Model Plant)

>gi|281199944|gb|GU223224.1| Pisum sativum sulfiredoxin precursor protein

(Srx) gene, complete cds

ATGGCGGCGAGCAACTTTCTGCTGCAGCTGCCGCTGCGCAGCTTTACCGTGATTAACGTGGCGAGCGCGA

GCAGCAGCAACGGTTCGCCGCCGGTGATCGGAGGATCTAGCGGCGGTGTAGGACCGATGATTGTGGAATT

ACCGTTGGAGAAGATACGAAGACCGTTGATGCGAACCAGATCCAACGATCAGAACAAAGTGAAAGAGCTT

ATGGATAGTATCCGTCAAATCGGTCTTCAAGTTCCGATTGATGTGATTGAAGTTGATGGAACTTACTATG

GGTTCTCGGGATGTCACAGATACGAGGCGCATCAGAAGCTAGGGCTTCCAACTATACGTTGCAAAATCCG

TAAAGGAACAAAGGAAACATTAAGGCATCATCTTCGCTGA

2. Pisum sativum (Target Plant)

>gi|326937563|emb|FN691474.1| Arabidopsis thaliana sph6 gene for S Protein

Homologue 6

ATGAATTCGTCTAACATAAATTTCCTAACAATTTTCTACTCAATGTTTATAATCATCTTTATAGTATTAA

TATCTTTGATAGGCTGTGAAACTCTACAACATGATGGAAAAGTATTTCCAATGAAAGGTCCTCTTACTAG

GGTTGTGATTTATAATGACAATGATTATCTTTTAGGAGTTCATTGTAAATCAAGAGATGATGATCATGGC

TTCCATATTCTACAAAAAGGTGGATTATATGGTTGGATGTTTTACGTGAATTTTATGAATTCGACACTCT

ACTTCTGTGGATTTAGCCAAGAACAAGTAAAAAAAGGTGTGTTCGATATTTATAAAGCGGTTAGAGATTC

TTCTAGATGTAGAAATTGTACTTGGGAAGCAAAGGAAGATGGTATTTATGGATATGGCGAGATTCCTAAG

AAAAATCCTTTGTTTTATAAGTGGCTAATGTAA

References:

1. Hall, T.A. 1999. BioEdit: a user-friendly biological sequence alignment editor andanalysis program for Windows 95/98/NT. Nucl. Acids. Symp. Ser. 41:95-98.

Principle Outcomes:DNA molecule: arabidpsis thaliana

Length = 453 base pairsMolecular Weight = 136212 Daltons, single stranded

Molecular Weight = 273979 Daltons, double stranded


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G+C content = 31.35%

A+T content = 68.65%

Nucleotide Number Mol%

A 150 33.11

C 55 12.14

G 87 19.21

T 161 35.54

DNA molecule: pisum sativum

Length = 390 base pairs

Molecular Weight = 118241 Daltons, single stranded

Molecular Weight = 237117 Daltons, double stranded

G+C content = 50.00%

A+T content = 50.00%

Nucleotide Number Mol%

A 108 27.69

C 83 21.28

G 112 28.72

T 87 22.31


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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.


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Experiment No.5

Aim: To identify the Drug targets in plants.

Principle:

Molecular modelling encompasses all theoretical methods and computational techniquesused to model or mimic the behaviour ofmolecules. The common feature of molecular

modelling techniques is the atomistic level description of the molecular systems; 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. Docking is frequently used to predict the

binding orientation ofsmall molecule drug candidates to their protein targets in order to in

turn predict the affinity and activity of the small molecule.


Receptor: 3O74

Ligand: 2XZP

Tool: HEX6.3

Reference: Lupas, A., Van Dyke, M., and Stock, J. (1991) Predicting Coled Coils from

Protein Sequences, Science 252:1162-1164
http://en.wikipedia.org/wiki/Model_(abstract)http://en.wikipedia.org/wiki/Moleculehttp://en.wikipedia.org/wiki/Binding_(molecular)http://en.wikipedia.org/wiki/Supramolecular_chemistryhttp://en.wikipedia.org/wiki/Small_moleculehttp://en.wikipedia.org/wiki/Drughttp://en.wikipedia.org/wiki/Drughttp://en.wikipedia.org/wiki/Small_moleculehttp://en.wikipedia.org/wiki/Supramolecular_chemistryhttp://en.wikipedia.org/wiki/Binding_(molecular)http://en.wikipedia.org/wiki/Moleculehttp://en.wikipedia.org/wiki/Model_(abstract)


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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


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Experiment No.6

Aim: To design plant nucleotide primer using primer designing tool.

Principle:

A primer is a strand of nucleic acid that serves as a starting point for DNA synthesis. Theyare required for DNA replication because the enzymes that catalyze this process, DNA

polymerases, can only add new nucleotides to an existing strand of DNA. The polymerase

starts replication at the 3'-end of the primer, and copies the opposite strand.

In most cases of natural DNA replication, the primer for DNA synthesis and replication is a

short strand of RNA


Sequence: Arabidopsis thaliana actin 3 gene, complete cds. GenBank: U39480.1

Tool: Geneious Pro 5.3.5

Reference:

Drummond AJ, Ashton B, Buxton S, Cheung M, Cooper A, Duran C, Field M, Heled J,

Kearse M, Markowitz S, Moir R, Stones-Havas S, Sturrock S, Thierer T, Wilson A (2011)

Geneious v5.4, Available from http://www.geneious.com/

Principle Outcome:
http://www.geneious.com/http://www.geneious.com/


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Type Name Sequence Minimum Maximum Length #

Intervals

Direction

primer_bind 1st forwardprimer

TGAGCAGGAGCTTGAGACGGC 2501 2521 21 1 forward

primer_bind 2nd forwardprimer

GCCTTAACCGAGGCCGAGCG 848 867 20 1 forward

primer_bind 3rd forward

primer


primer_bind 4th forward

primer


primer_bind 5th forward

primer


primer_bind_reverse 1st reverse

primer

ACCTCAGGGCAACGGAAACGC 2591 2611 21 1 reverse

primer_bind_reverse 2nd reverseprimer

GCTTCGAATTCGGCACACCTCGT 933 955 23 1 reverse

primer_bind_reverse 3rd reverse

primer

GCTTCGAATTCGGCACACCTCG 934 955 22 1 reverse

primer_bind_reverse 4th reverse

primer

GGCTTCGAATTCGGCACACCT 936 956 21 1 reverse

primer_bind_reverse 5th reverse

primer

AGGCTTCGAATTCGGCACACC 937 957 21 1 reverse

Conclusion:

We have successfully designed primers for Arabidopsis thaliana actin 3 gene, complete cds

sequence using primer designing tool Geneious Pro 5.3.5


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Experiment No.7

Aim: To draw phylogram using different plant protein sequence.

Principle: Phylogenetic methods can be used for many purposes, including analysis of

morphological and several kinds of molecular data.

Method specification:

Plants:1. oryza sativa2. Pseudoterranova decipiens and3. solanum tuberosum

Tool: Geneious 5.1.7

Principle Outcomes:


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Tree:

+ oryza sativa

|

+==========================================================================

========================= Pseudoterranova decipiens

|+=============================== solanum tuberosum

In Newick format:

('oryza sativa':0.0,'Pseudoterranova decipiens':1.242189,'solanum

tuberosum':0.3923650000000001);

Conclusion: we have successfully drawn phylogram using plant protein sequences of oryza

sativa, Pseudoterranova decipiens and solanum tuberosum


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Experiment No.8

Aim: To identify the secondary metabolites as drug targets for diseases in plants.

Principle:

Secondary metabolites are organic compounds that are not directly involved in thenormal growth, development, or reproduction of an organism. Secondary metabolites often

play an important role in plant defense against herbivory and other interspecies defenses.

Plants use secondary metabolites as medicines, flavorings, and recreational drugs. Secondary

metabolites are essentially low molecular weight compounds, sometimes having complex

structures. They function in processes as diverse as immunity, anti-herbivory, pollinator

attraction, communication between plants, maintaining symbiotic associations with soil flora,

enhancing the rate of fertilization etc., and hence are significant from the evo-devo

perspective


Receptor: 2XAM (Plant Hydrocarbon)

Ligand: 2XAL

Tool: HEX6.3

Reference: Lupas, A., Van Dyke, M., and Stock, J. (1991) Predicting Coled Coils from

Protein Sequences, Science 252:1162-1164

Principle Outcome:
http://en.wikipedia.org/wiki/Organic_compoundhttp://en.wikipedia.org/wiki/Cell_growthhttp://en.wikipedia.org/wiki/Biological_developmenthttp://en.wikipedia.org/wiki/Reproductionhttp://en.wikipedia.org/wiki/Reproductionhttp://en.wikipedia.org/wiki/Biological_developmenthttp://en.wikipedia.org/wiki/Cell_growthhttp://en.wikipedia.org/wiki/Organic_compound


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Conclusion:

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

Msc tics Practical.

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