Genetic basis, biosynthetic pathways and molecular …...evolution by linking genes, chemicals,...
1
Kipling Will, UC Berkeley Wendy Moore, U. of Arizona Aman Gill, UC Berkeley Tanya Renner, San Diego State U. Athula Attygalle, Stevens Inst. of Tech. Funding from the National Science foundation DEB1556957 DEB1556813 DEB1556931 DEB1556898 The Evolution of Geadephagan Chemical Defense 1. Gland cells I. TISSUE PREPARATION 2. Whole body (minus gland cells) II. HIGH-THROUGHPUT SEQUENCING Sequence tens of billions of base pairs in 250 bp paired-end reads adaptor adaptor Forward Reverse 250 bp 250 bp 1. Three transcriptomes from gland cells III. TRANSCRIPTOME ASSEMBLY contig 1 ≥500 bp IV. GENE ONTOLOGY ANNOTATION V. RNASEQ ANALYSES contig 1 contig 2 GO terms Biological Process Molecular Function Cellular Component GO terms Biological Process Molecular Function Cellular Component adaptor adaptor Forward Reverse 250 bp 250 bp 2. Three transcriptomes from whole body x3 x3 contig 2 ≥500 bp Cells 1 Cells 2 Cells 3 Body 1 Body 2 Body 3 Gland-speci c transcript STEP 2 contig 1 contig 2 high low tinyurl.com/zfeqj52 Check out the KQED DeepLook video about our NSF research. Genetic basis, biosynthetic pathways and molecular evolution of carabid quinone production Carabid beetles are the largest clade of organisms to use a single homologous gland system to produce at least 18 distinct classes of defensive chemical compounds. Quinone production: independently evolved or ancestrally conserved? Chemical assays . Beetles are injected with labelled chemical precursors that are incorporated into gland products during synthesis. Using GC-MS the labelled products are analyzed to infer biosynthetic pathways. Steps in the pathways suggest enzymes likely to be involved, clarifying the evolution of biosynthesis at the chemical level and guiding the search for genes underlying quinone production. What biosynthetic pathways and which genes are used for quinone production? The chemical composition and delivery mechanisms of carabid defensive compounds are well understood, but little is known about the biosynthetic pathways, genetic architecture, and evolutionary history underlying their production. Transcriptome sequencing. Gland-only transcriptomes are compared to transcriptomes from whole bodies (minus gland) to identify genes expressed in secretory gland cells during chemical synthesis.Transcripts are functionally annotated and assigned to known metabolic pathways.This information is combined with expression patterns, biosynthesis assays, and data from Tribolium and other systems to identify candidate genes critical for quinone production. Phylogenetic analysis. four pairs of species, each representing one of four lineages of among all bombardiers.This will allow us to examine the extent of independent versus conserved evolution, identify possible patterns of convergence and exaptation, and infer the evolutionary history of quinone production, e.g. birth-death dynamics, patterns of selection, and variation in evolutionary rates. Functional validation of candidate genes using RNAi Functional assays. Candidate gene expression is knocked down using gene silencing (RNAi) and the resulting gland products are examined, identifying experimentally validated genes with quinone-less or quinone-limited RNAi phenotypes. In a pilot study on Brachinus , knock down of three transcripts reduced expression and resulted in reduced quantities of quinones. Authors & Funding some Clivinini some Chlaenini Charles Hedgcock, (c) 2015 Wendy Moore evolution by linking genes, chemicals, biosynthetic pathways and phylogeny in carabid beetles. We aim to develop an understanding of defensive chemical RNAi knockdown of three Brachinus genes. based on homology with two genes known to be essential to quinone production in Tribolium castaneum . Knockdown of genes resulted in reduced expression measured via qPCR and absence of defensive toluquinones. ARSB = Arylsulfatase B; MDRP = multi-drug resistance protein, isoform 1 and 2. !"!! !"#$ !"$! !"%$ &"!! !"!!!! !"!!%! !"!&'! !"!#&! !"!#(! !)*!! &)+!' #)+!' ,)+!' ')+!' ARSB-1 MDRP-1 MDRP-2 Toluquinone level RNAi Control Gene expression Relative mRNA level Seeking graduate students to study carabid chemical defense evolution! Paussinae Bombardiers Brachininae Bombardiers m-cresol toluhydroquinone toluquinone tinyurl.com/hlq99ru Carabidae Other beetles Quinones Formic acid Other chemical classes !
Transcript of Genetic basis, biosynthetic pathways and molecular …...evolution by linking genes, chemicals,...
Kipling Will, UC BerkeleyWendy Moore, U. of ArizonaAman Gill, UC BerkeleyTanya Renner, San Diego State U.Athula Attygalle, Stevens Inst. of Tech.
Funding from the National Science foundationDEB1556957 DEB1556813 DEB1556931 DEB1556898
The Evolution of Geadephagan Chemical Defense
1. Gland cells
I. TISSUE PREPARATION2. Whole body (minus gland cells)
II. HIGH-THROUGHPUT SEQUENCING
Sequence tens of billions of base pairs in 250 bp paired-end reads
adaptor
adaptor
Forward Reverse250 bp 250 bp
1. Threetranscriptomes from gland cells
III. TRANSCRIPTOME ASSEMBLY
contig 1 ≥500 bp
IV. GENE ONTOLOGY ANNOTATION
V. RNASEQ ANALYSES
contig 1
contig 2
GO termsBiological ProcessMolecular FunctionCellular Component
GO termsBiological ProcessMolecular FunctionCellular Component
adaptor
adaptor
Forward Reverse250 bp 250 bp
2. Three transcriptomes from whole body
x3 x3
contig 2 ≥500 bp
Cells 1 Cells 2 Cells 3 Body 1 Body 2 Body 3
Gland-speci c transcript
STEP 2
contig 1contig 2
highlow
tinyurl.com/zfeqj52
Check out the KQED DeepLook video about our NSF research.
Genetic basis, biosynthetic pathways and molecular evolution of carabid quinone production
Carabid beetles are the largest clade of organisms to use a single homologous gland system to produce at least 18 distinct classes of defensive chemical compounds.
Quinone production: independently evolved or ancestrally conserved?
Chemical assays . Beetles are injected with labelled chemical precursors that are incorporated into gland products during synthesis. Using GC-MS the labelled products are analyzed to infer biosynthetic pathways. Steps in the pathways suggest enzymes likely to be involved, clarifying the evolution of biosynthesis at the chemical level and guiding the search for genes underlying quinone production.
What biosynthetic pathways and which genes are used for quinone production?
The chemical composition and delivery mechanisms of carabid defensive compounds are well understood, but little is known about the biosynthetic pathways, genetic architecture, and evolutionary history underlying their production.
Transcriptome sequencing. Gland-only transcriptomes are compared to transcriptomes from whole bodies (minus gland) to identify genes expressed in secretory gland cells during chemical synthesis. Transcripts are functionally annotated and assigned to known metabolic pathways. This information is combined with expression patterns, biosynthesis assays, and data from Tribolium and other systems to identify candidate genes critical for quinone production.
Phylogenetic analysis. four pairs of species, each representing one of four lineages of
among all bombardiers. This will allow us to examine the extent of independent versus conserved evolution, identify possible patterns of convergence and exaptation, and infer the evolutionary history of quinone production, e.g. birth-death dynamics, patterns of selection, and variation in evolutionary rates.
Functional validation of candidate genes using RNAiFunctional assays. Candidate gene expression is knocked down using gene silencing (RNAi) and the resulting gland products are examined, identifying experimentally validated genes with quinone-less or quinone-limited RNAi phenotypes. In a pilot study on Brachinus, knock down of three transcripts reduced expression and resulted in reduced quantities of quinones.
Authors & Funding
some Clivinini
some Chlaenini
Charles Hedgcock, (c) 2015 Wendy Moore
evolution by linking genes, chemicals, biosynthetic pathways and phylogeny in carabid beetles.
We aim to develop an understanding of defensive chemical
RNAi knockdown of three Brachinus genes. based on homology with two genes known to be essential to quinone production in Tribolium castaneum. Knockdown of genes resulted in reduced expression measured via qPCR and absence of defensive toluquinones. ARSB = Arylsulfatase B; MDRP = multi-drug resistance protein, isoform 1 and 2.
ARSB-1
!"!!
!"#$
!"$!
!"%$
&"!!
MDRP-2
!"!!!!
!"!!%!
!"!&'!
!"!#&!
!"!#(!
MDRP-1
!)*!!
&)+!'
#)+!'
,)+!'
')+!'
Control RNAi
rela
tive
mRN
A le
vel
toluquinone ++ - - ++ - - ++ - -
ARSB-1 MDRP-1 MDRP-2
Toluquinonelevel
RNAiControlGene expression
Rela
tive
mRN
A le
vel
Seeking graduate students to study carabid chemical
defense evolution!
Paussinae
Bombardiers
Brachininae
Bombardiers
m-cresol toluhydroquinone toluquinone
tinyurl.com/hlq99ru
Carabidae
Otherbeetles
Quinones
Formic acid
Other chemical classes
!
