Roy J. Carver Department of Biochemistry, Biophysics & Molecular Biology
Transcriptomics analysis reveals shared precursors in
the biosynthesis of hydrocarbons in photosynthetic
green algae and plants
Adarsh Jose
BCB/BBMB
Iowa State University
Major Professor
Dr. Basil J. Nikolau
Dr.Wenmin Qin did all the wet lab work !
Simple hydrocarbons
Linear n-alkanes
Linear n-alkenes
2
Plant Lipids: Structure, Metabolism & Function
(Gordon Research Seminar)
3
Plant Lipids: Structure, Metabolism & Function
(Gordon Research Seminar)
Four hydrocarbon producing organisms
4
Plant Lipids: Structure, Metabolism & Function
(Gordon Research Seminar)
Emiliania huxleyi
Plant Lipids: Structure, Metabolism & Function
(Gordon Research Seminar)
Botryococcus braunii
6
Plant Lipids: Structure, Metabolism & Function
(Gordon Research Seminar)
Zea Mays B-73 Silk emerged from
and encased in the husk
Upper and lower surface of Pisum Sativum leaves
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Plant Lipids: Structure, Metabolism & Function
(Gordon Research Seminar)
Hydrocarbon(umol/g)
Hydrocarbon (umol/g)
30
B.Braunii
25
20
15
10
5
0
NP+/C-
NP-/C+
6
E.hux-1516
5
4
3
2
1
0
NP-/C+
NP+/C-
upper
lower
10%
8%
6%
4%
2%
B17_2 B17 B15_2 B15 B13_2 B13 B11_2 B11 B9_2 B9
B7
B5
Hydrocarbon content of leaf epidermis
(older branches to the right)
0%
6
Hydrocarbon (umol/g)
Hydrocarbon content
(g/g dry weight)
12%
Corn Silks
5
4
3
2
1
0
-4
-3
-2
-1
1
2
3
Segmentation of Corn Silk
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Plant6 Lipids:
Metabolism & Function
Days Structure,
post emergence
8
(Gordon Research Seminar)
fastx toolkit
Illumina PE
Transcriptome raw data
Quality filter
Trinity Assembler
Pool reads and
Assemble Transcriptome
Reference Transcriptome
Genome sequence
available ?
No
Yes
Reference genome
Reference Sequences
Functional Annotations from
the Genome Annotation
BLASTX
Map PE Reads to the Reference
Sequences
Uniprot-KB,
Conserved Domain Database,
Sequenced Phylogenetic Neighbors
Estimate Normalized Read
Counts
Tophat PE Aligner /
Bowtie PE Aligner
CuffLinks as FPKM /
RSEM as FPKM
GO Enrichment - topGO
Fisher Exact Test, EASE Score
Statistical Analysis –
List Enrichment
Statistical Analysis –
Diff Exp
BLASTX
Global Results
Diff Exp usind Cuffdiff /
DESeq R package
Curated Protein Sequences
Pathway Mapping
Plant Lipids: Structure, Metabolism & Function
(Gordon Research Seminar)
Query with genes from carbon fixation, gluconeogenesis,
glycolysis, TCA cycle, fatty-acid synthesis, elongation and
hydrocarbon synthesis TAIR and Aralip databases
Chlamydomonas reinhardtii
Protein models
Handpicked from other
organisms
Denovo assembled contigs/
Gene Models
BLASTX
Heuristics using BLAST
e-value
Summarize Expression data
for each Enzyme/Transporter
Plant Lipids: Structure, Metabolism & Function
(Gordon Research Seminar)
30
20
30
Waris with CO2
10
B3N without CO2
0
0
2
4
6
8
10
Time (days)
Hydrocarbon (umol/g)
Total hydrocarbons (μmol/g dry weight)
A case study - Botryococcus. Braunii (UTEX 572)
25
20
15
10
5
0
NP+/C-
NP-/C+
Waris medium: Low nutrients(NO3-PO4-), high C(HCO3-)
Modified B3N medium: High nutrients(NO3-PO4-), low C(HCO3-)
11
Plant Lipids: Structure, Metabolism & Function
(Gordon Research Seminar)
12
Plant Lipids: Structure, Metabolism & Function
www.pathvisio.org
(Gordon
Research Seminar)
13
Plant Lipids: Structure, Metabolism & Function
www.pathvisio.org
(Gordon
Research Seminar)
Summarizing Results from all four systems
Plant Lipids: Structure, Metabolism & Function
14
(Gordon Research Seminar)
Order of systems in the boxes
Corn Silk
Pea Leaves
E huxleyi
B braunii
15
Plant Lipids: Structure, Metabolism & Function
www.pathvisio.org
(Gordon
Research Seminar)
Order of systems in the boxes
Corn Silk
Pea Leaves
E huxleyi
B braunii
16
Plant Lipids: Structure, Metabolism & Function
www.pathvisio.org
(Gordon
Research Seminar)
Take away points
•
•
•
Four evolutionary diverse organisms were
compared for shared metabolic pathways
synthesizing hydrocarbons and their precursors.
They seem to share several steps in the
pathways and diverge at some.
Key enzymes which could act as potential break
points are expressed highly.
17
Plant Lipids: Structure, Metabolism & Function
(Gordon Research Seminar)
Acknowledgements
•
•
•
Dr. Wenmin Qin
Dr. Marna Yandeau-Nelson
Dr. Basil J. Nikolau
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Plant Lipids: Structure, Metabolism & Function
(Gordon Research Seminar)