Derrick J. Reynolds and Dr. P. Jeff Maughan, Plant and Animal Sciences

Quinoa (Chenopodium quinoa) is an important crop for subsistence farmers in the Altiplano (high plains) of Peru, Bolivia, and Argentina. Quinoa’s seed is highly nutritious, but the seed coat of quinoa contains several anti-nutritive triterpenoid saponin compounds. The biological role of saponins in plants is not clear, however saponin is suggested to act as an anti-microbial and anti-feedant and are known to inhibit nutrient uptake in humans. The saponins of the Caryophyllaceous family, such as quinoa, are almost completely based on β-amyrin. Indeed, the first committed step in the pathway towards saponins biosynthesis is the cyclization of 2,3-oxidosqualene by β-amyrin synthase. Unfortunately, apart from a few structural prerequisites, little else is known about the saponin biogenesis in quinoa. Previously, we showed that the bitter saponin production in quinoa is controlled by a single dominant gene (BSP gene) and have placed the BSP locus on the genetic marker based linkage map. Since β-amyrin synthase is the first committed step in saponin biosynthesis, we have cloned and characterized the β-amyrin synthase gene from quinoa.

In previous work I identified a 1400 basepair partial sequence of the β-amyrin synthase gene in quinoa. This was accomplished by extracting and the reverse transcription of mRNA from immature quinoa (‘NL-6’) seeds at different stages of development (16, 24, 32, 40 days post-anthesis). Degenerate oligos derived from conserved regions of β-amyrin synthase genes from soybean and Arabidopsis were used to amplify fragments of the β-amyrin synthase gene from quinoa. These amplified fragments were cloned and sequenced. A BLASTX analysis indicated that cloned fragments showed extensive homology with other β-amyrin synthase genes.

The full length sequence of the β-amyrin synthase gene was elucidated using the Rapid Amplification of cDNA Ends (RACE) technique. RNA was extracted and reverse transcribe to cDNA from quinoa accession ‘NL-6’ seeds at 24 post-anthesis. Gene Specific Primers derived from the 1400 bp internal fragment were used in concert with the 5’ and 3’ adapter-based primers to amplify the 5’ and 3’ ends of the β-amyrin synthase. The amplified fragments were then cloned and sequenced.

Using the RACE method a full-length cDNA sequence with high homology to the β-amyrin synthesis gene was obtained. The open reading frame consists of 2,292 nucleotides encoding a protein of 763 amino acids predicted to weighs 87.85 kD. A BLASTX homology analysis of the amino acid sequence revealed 85%, 79%, and 70% identity with β-amyrin synthase of Vaccaria hispanica (ABK76265), Medicago truncatula (AJ430607), and Arabidopsis thaliana (NP_683508) respectively. The β-amyrin synthase gene contains a DCTAE motif which is thought to be part of the active site of oxidosqualene cyclase and a Typ residue in the MWCYCR motif which has been noted to play a role in the protein’s formation.A phylogenetic analysis of the AA sequence grouped cqBAS in the same clade (although distantly) with V. hisparica and G. pariculata, the only two other sequences from the family Caryophyllaceae.

After the β-amyrin synthase gene has been identified, it will be possible to co-segregate the presence or absence of the β-amyrin synthase gene with the presence or absence of saponin deposition in quinoa seeds determined by genetic mapping in a segregating F2 quinoa population to verify that the β-amyrin synthase gene indeed codes for a protein essential in bitter saponin biosyntheses.

There is evidence of a second homologous locus for the β-amyrin synthase gene. This is very possible given that quinoa is an allotetraploid. We hope to conclusively identify the presence or absence of a second allele.

In retrospect this project was more difficult and of a much larger scope than I had initially realized. However, the experience I have gained, as a result of working with this project has been a great way to learn about cutting-edge technologies in areas related to my fields of study. It has also been an excellent opportunity to make connections that will enable me to take the next step in my academic and professional careers with confidence. I also believe that it has provided me with tools to enable the realization of my personal goals in life and science.

References

• A genetic linkage map of quinoa (Chenopodium quinoa) based on AFLP, RAPD, and SSR markers P. J. Maughan et al. Theor Appl Genet (2004) 109: 1188–1195
• Biosynthesis of Triterpenoid Saponins in Plants Kosmas Haralampidis, Miranda Trojanowska and Anne E. Osbourn Advances in Biochemical Engineering/Biotechnology, Vol. 75 (2002)
• Genetic Mapping of the Bitter Saponin Production Locus (BSP Locus) in Chenopodium quinoa Willd Marc D. Ricks, P. Jeffery Maughan, Brigham Young University Library Reserve (2005)