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 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 cross- referencing the DNA sequence produced with the known cDNA sequence of the gene. Introduction Carrots have various carotenoid pigments, which are responsible for the colors red, yellow, and orange. In humans, when certain carotenoid pigments are taken in, they are converted to vitamin A (such as β-carotene, α-carotene, and cryptoxanthin) or serve as antioxidants (such as lutein) (Kubler, 1963). Lutein strengthens the eyes of humans and can decrease the risk for eye diseases that affect the macula (Johnson 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 CHXE (Tian et al., 2003). A bacterial artificial chromosome (BAC) library consists of Escherichia coli bacteria that have a section of another organism's DNA spliced into its own (Shizuya, 1992). 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 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 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 be found within the genome map (Just et al., 2007). Along with finding the location 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. Materials and Methods This experiment is in association with Dr. P.W. Simon of the Dept. of Horticulture, UW-Madison. Three BACs in a carrot BAC library of 110,000 colonies that have already been created, screened, and identified to have the CHXE gene in them will be used to determine the gene’s genomic sequence. The BACs that were studied are located in plates A206 well F1, B212 well E20, and B212 well F21 in the 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, 5’CDSendR/LUT1-5RACE, LUT1-5’RACE/CHXE-1523R, and CHXE-1461F/CHXE- 3UTRendR. Sequencing PCR reactions were then performed and the products were cleaned up using the magnetic bead clean up technique. The cleaned products were then submitted to the University of Wisconsin Biotechnology center (Madison, WI) for sequencing. Results and Discussion The research for this experiment has not been completed yet. The cDNA sequence of the CHXE gene 1803 base pairs long, but the results from the PCR reactions 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 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). 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. References 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 (2007): Kubler Werner “Carotin als Provitamin A beim Menschen.” Deutsche Medizinische Wochenschreiben 88 (1963): 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): 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): Tian, Li, et al. “Functional analysis of beta- and epsilon-ring carotenoid hydroxylases in Arabidopsis.” Plant Cell 6 (2003): CHXE FINAL SEQ F TO CHXE FINAL SEQ R CHXE- 5'UTRendF TO CHXE- 3'UTRend R CHXE- 5'UTRend F to LUT1- 5'RACE LUT1- 5RACE TO CHXE- 3'UTRendR CHXE FINALSE Q F TO LUT1- 5'RACE LUT1- 5RACE TO CHXE FINALSE Q R CHXE- 5'UTRendF TO CHXE- 5'CDSend R CHXE- 5CDSendR TO CHXE- 3'UTRend R CHXE FINALSEQ F TO CHXE- 5'CDSendR CHXE- 5CDSendR TO CHXE FINALSEQ R CHXE- 5'UTRendF TO CHXE3'endR1 CHXE FINALSEQ F TO CHXE3'endR1 CHXE-contig- FULL F TO CHXE- 5'CDSendR CHXE- 5CDSendR TO LUT1-5RACE CHXE- contig- FULL F TO CHXE 5CDSendR CHXE- contig-FULL F TO LUT1- 5'RACE Figure 1 – Testing of primer pairs with BAC A206F1 using Fermentas 1kb gene ruler to measure size LUT1-5'RACE TO CHXE- 3UTRendR LUT1-5'RACE TO CHXE FINAL SEQ LUT1-5'RACE TO CHXE- 1523R CHXE-1461F TO CHXE- 3UTRendR CHXE-1461F TO CHXE FINAL SEQR Figure 2 – Testing of primer pairs with BAC A206F1 using Fermentas 1kb gene ruler to measure size 1000bp 1500bp 2000bp 2500bp 3000bp 3500bp 4000bp 5000bp 6000bp 8000bp 10000bp CHXE FINALSEQ F TO 5'CDSend R 5CDSend R TO LUT1- 5’RACE LUT1- 5RAC E TO CHXE- 1523R CHXE- 1461F TO CHXE 3UTRen dR 250bp 500bp 750bp 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. Figure 3 – PCR Gel of unsuccessful PCR reaction for BAC B212E20 Figure 4 – PCR Gel of unsuccessful PCR reaction for BAC B212F21