Isolating and Sequencing the Aconitase-4 Gene
Mentor 1
Devinder Sandhu
Location
Union Wisconsin Room
Start Date
24-4-2015 10:30 AM
End Date
24-4-2015 11:45 AM
Description
Isozymes are variants of the same enzyme that differ in sequence but catalyze the same chemical reaction. Aconitase isozymes catalyze the interconversion of the three tricarboxylic acids: citrate, cis-aconitate, and isocitrate in the Krebs cycle. The Aconitase-4 isozyme has been used in mapping studies in soybean and has been used to study allele switching. For the study, parent plants BSR 101 and PI 290136 were crossed. The F2 generation was scored for Aconitase-4 (Aco-4) alleles and was used to make two bulks: one displaying the BSR 101 allele pattern, and one displaying the PI 290136 allele pattern. The bulks were then used in bulked segregant analysis (BSA), and tested with 700 primers. The gene was determined to be closest to Satt509, a marker located on Molecular linkage group (MLG) B1 (Chromosome 11). All the polymorphic MLG B1 primers were used on entire F2 population and genetic linkage map was developed. Aco-4 was mapped to a ~292kb region with BARCSOYSSR_11_323 and BARCSOYSSR_11_336 flanking the gene. In this region, there are 40 predicted genes. Glyma11g080600 is the most likely candidate as it shares sequence similarity to an aconitase gene. We have designed long range PCR primers to amplify Glyma11g080600 from two variants. We are in the process of sequencing Glyma11g080600. Sequence comparison may reveal critical differences between two isozyme variants and confirm isolation of the Aco-4 gene. /
Isolating and Sequencing the Aconitase-4 Gene
Union Wisconsin Room
Isozymes are variants of the same enzyme that differ in sequence but catalyze the same chemical reaction. Aconitase isozymes catalyze the interconversion of the three tricarboxylic acids: citrate, cis-aconitate, and isocitrate in the Krebs cycle. The Aconitase-4 isozyme has been used in mapping studies in soybean and has been used to study allele switching. For the study, parent plants BSR 101 and PI 290136 were crossed. The F2 generation was scored for Aconitase-4 (Aco-4) alleles and was used to make two bulks: one displaying the BSR 101 allele pattern, and one displaying the PI 290136 allele pattern. The bulks were then used in bulked segregant analysis (BSA), and tested with 700 primers. The gene was determined to be closest to Satt509, a marker located on Molecular linkage group (MLG) B1 (Chromosome 11). All the polymorphic MLG B1 primers were used on entire F2 population and genetic linkage map was developed. Aco-4 was mapped to a ~292kb region with BARCSOYSSR_11_323 and BARCSOYSSR_11_336 flanking the gene. In this region, there are 40 predicted genes. Glyma11g080600 is the most likely candidate as it shares sequence similarity to an aconitase gene. We have designed long range PCR primers to amplify Glyma11g080600 from two variants. We are in the process of sequencing Glyma11g080600. Sequence comparison may reveal critical differences between two isozyme variants and confirm isolation of the Aco-4 gene. /
