Transcript My CHXE Carrot BAC Research Poster

Sequencing and Sub-cloning of the CHXE gene in a Carrot (Daucus
carota) Bacterial Artificial Chromosome
Eric Victor¹, Dr. Pablo Cavagnaro², Dr. Doug Senalik², and Dr. Philipp W. Simon²
¹Biology 152 Independent Research Student, ²USDA-ARS, Vegetable Crops Research Unit, Department of Horticulture, University of Wisconsin-Madison, WI 53706.
CHXE FINAL
SEQ F TO
CHXE FINAL
SEQ R
Abstract
A Carrot (Daucus carota) bacterial artificial chromosome was cloned and the
plasmid DNA extracted from it. The DNA was tested for the presence of the CHXE
gene through PCR amplification utilizing various primers that spanned the entire
known cDNA sequence of the gene. Gel electrophoresis was performed on the
PCR products in a 0.7% agarose gel to determine whether the amplification was
successful and to determine the size of the different sections of the gene. The
amplified products were then cleaned using the Exo-Sap procedure to reamplify the
products to be submitted for gene sequencing at the UW Biotechnology center. The
sequences were returned and examined for the completion of the full length
genomic sequence. Introns within the gene were able to be mapped by crossreferencing the DNA sequence produced with the known cDNA sequence of the
gene.
CHXECHXE5'UTRendF 5'UTRend
TO CHXE- F to
3'UTRend LUT1R
5'RACE
LUT1CHXE
5RACE TO FINALSE
CHXEQ F TO
3'UTRendR LUT15'RACE
LUT15RACE
TO CHXE
FINALSE
QR
CHXECHXE5'UTRendF 5CDSendR
TO CHXE- TO CHXE5'CDSend 3'UTRend
R
R
CHXE5'UTRendF TO
CHXE3'endR1
CHXE
CHXE-contigFINALSEQ F TO FULL F TO
CHXE3'endR1
CHXE5'CDSendR
CHXE5CDSendR TO
LUT1-5RACE
CHXEcontigFULL F TO
CHXE
5CDSendR
CHXEcontig-FULL
F TO LUT15'RACE
studied are located in plates A206 well F1, B212 well E20, and B212 well F21 in the
LUT1-5'RACE
TO CHXE3UTRendR
LUT1-5'RACE LUT1-5'RACE
TO CHXE
TO CHXEFINAL SEQ
1523R
CHXE-1461F
TO CHXE3UTRendR
CHXE-1461F
TO CHXE
FINAL SEQR
Figure 1 – Testing of primer pairs with BAC A206F1 using Fermentas
1kb gene ruler to measure size
Simon lab (Madison, WI). The BAC DNA was isolated as a small-scale culture and
the DNA extracted from it. PCR reactions were performed on the BACs to determine
which combination of primers would allow for the sequencing of the genomic
sequence (Fig. 1,2). Four primer pairs were selected: CHXE-FinalSeq F/ 5CDSendR,
CHXE
FINALSEQ
F TO
5'CDSend
R
5CDSend
R TO
LUT15’RACE
LUT15RAC
E TO
CHXE1523R
5’CDSendR/LUT1-5RACE, LUT1-5’RACE/CHXE-1523R, and CHXE-1461F/CHXE-
CHXE1461F
TO
CHXE
3UTRen
dR
3UTRendR. Sequencing PCR reactions were then performed and the products were
cleaned up using the magnetic bead clean up technique. The cleaned products were
Figure 2 – Testing of primer pairs with BAC
A206F1 using Fermentas 1kb gene ruler to
measure size
then submitted to the University of Wisconsin Biotechnology center (Madison, WI) for
sequencing.
250bp
humans and can decrease the risk for eye diseases that affect the macula (Johnson
Results and Discussion
500bp
et al., 2000). This nutritional fact makes lutein an important element in determining
whether a carrot purchased at the store would be healthier for the consumer. Lutein
is synthesized by the enzyme ε-ring carotene hydroxylase. The gene that codes for
ε-ring carotene hydroxylase in the biosynthetic pathway of carotenoids is called
The research for this experiment has not been completed yet. The cDNA sequence
750bp
of the CHXE gene 1803 base pairs long, but the results from the PCR reactions
1000bp
show that the genomic sequence is over 6400 base pairs (Fig. 5). This increase in
size between the two types of sequences is due to the possibility of the gene
CHXE (Tian et al., 2003).
1500bp
A bacterial artificial chromosome (BAC) library consists of Escherichia coli bacteria
2000bp
2500bp
3000bp
3500bp
4000bp
5000bp
6000bp
8000bp
10000bp
Figure 3 – PCR Gel of unsuccessful
PCR reaction for BAC B212E20
A carrot BAC library is a collection of E. coli colonies, each with a different piece of
the carrot genome spliced into its plasmids.
This experiment is in association with Dr. P.W. Simon of the Dept. of
them will be used to determine the gene’s genomic sequence. The BACs that were
serve as antioxidants (such as lutein) (Kubler, 1963). Lutein strengthens the eyes of
that have a section of another organism's DNA spliced into its own (Shizuya, 1992).
Materials and Methods
that have already been created, screened, and identified to have the CHXE gene in
Carrots have various carotenoid pigments, which are responsible for the colors red,
are converted to vitamin A (such as β-carotene, α-carotene, and cryptoxanthin) or
CHXE5CDSendR TO
CHXE
FINALSEQ R
Horticulture, UW-Madison. Three BACs in a carrot BAC library of 110,000 colonies
Introduction
yellow, and orange. In humans, when certain carotenoid pigments are taken in, they
CHXE
FINALSEQ F
TO CHXE5'CDSendR
This spliced-in DNA can than be
extracted from the E. coli when necessary. This is done so that the segments of
DNA from the same sample can be easily and continuously extracted and used to
perform tests and experiments. The bacteria are able to replicate themselves and
containing over 4600 intron base pairs. This increase in size of the PCR products is
making the sequencing of the gene more difficult to accomplish as new primers, as
well as primer pairs are going to be necessary to sequence these introns. Currently,
no successful PCR has been conducted that could be sent to the Biotechnology
center for sequencing. Research is continuing in trying to perform successful PCR
reactions for the other two sets of BAC DNA, B212E20 and B212F21 (Fig. 3,4).
create a large amount of identical DNA that can be extracted (Shultz, 2006).
In previous research, sequences and suspected map locations for various genes
within the carrot genome have been determined.
The various enzymes that
participate in the carotene biosynthetic pathway have been located and they will now
Figure 4 – PCR Gel of unsuccessful
PCR reaction for BAC B212F21
Figure 5 – PCR Gel showing length in base pairs of amplified DNA.
The entire CHXE gene is amplified using the four primer pairs with
minimal known overlap.
Just, Brian, et al. “Carotenoid biosynthesis structural genes in carrot (Daucus carota): isolation, sequence-characterization,
single nucleotide polymorphism (SNP) markers and genome mapping.” Theoretical and Applied Genetics 114-4
(2007): 693-704.
Kubler Werner “Carotin als Provitamin A beim Menschen.” Deutsche Medizinische Wochenschreiben 88 (1963): 1319-1325.
be found within the genome map (Just et al., 2007). Along with finding the location
Shizuya, Hiroaki, et al. “Cloning and stable maintenance of 300-kilo base-pair fragments of human DNA in Escherichia coli
using an F-factor-based vector.” Proceedings of the National Academy of Sciences 89 (1992): 8794-8797.
of the CHXE gene in the genome, genes that are located close to it will also be
looked for in order to complete the genomic map.
References
Acknowledgements
At this time I would like to thank Dr. Simon for his mentorship throughout this project, the use of his laboratory, his suggestions and editorial
input. I would like to thank Dr. Cavagnaro, Dr. Diaby, and Dr. Doug Senalik for their teachings in PCRs and the analysis of electrophoresis gels.
I would also like to thank Dr. Iovene for helping me with the growing of the BAC DNA.
Shultz, JL, et al. “Three minimum tile paths from bacterial artificial chromosome libraries of the soybean (Glycine max cv.
'Forrest'): tools for structural and functional genomics.” Plant Methods 2 (2006): 9-18.
Tian, Li, et al. “Functional analysis of beta- and epsilon-ring carotenoid hydroxylases in Arabidopsis.” Plant Cell 6 (2003): 132032.