Date of Graduation

8-2016

Document Type

Thesis

Degree Name

Master of Science in Cell & Molecular Biology (MS)

Degree Level

Graduate

Department

Biological Sciences

Advisor

Kenneth L. Korth

Committee Member

Sun-Ok Lee

Second Committee Member

David S. Mcnabb

Keywords

Biological sciences; Gene regulation; Legume; Specialized metabolism

Abstract

Saponins are a large family of specialized metabolites produced in many plants. They can have negative effects on a number of plant pests and are thought to play a role in plant defense. With current and possible future uses in industry and agriculture, saponins have also been shown to be hypocholesterolemic, hypoglycemic, immunostimulatory, antioxidative, anti-inflammatory, and cytotoxic. In spite of their usefulness, our understanding of the genetic basis for saponin biosynthesis is still incomplete. We generated recombinant populations with parents from genetically distinct accessions of Medicago truncatula, with either high or low accumulation and varying profiles of saponins. Primers for a PCR-based parental test were developed from single-nucleotide polymorphisms in the sequence encoding CYP72A68, a cytochrome P450 enzyme involved in the biosynthesis of M.truncatula sapogenins. Comparison of translated CYP72A68 amino acid sequences across accessions revealed high similarity, and comparison with CYP72A proteins from other plant species suggests similar protein functioning across the accessions. The F2 generation plants from one recombinant population were screened for hemolytic saponin accumulation levels in leaf extracts by measuring cleared zones in blood agar plates. Three distinct phenotypes were observed. Plants in the F2 generation showed either a high or low hemolysis phenotype matching those of the parent plants, or an intermediate level of hemolysis. The high:medium:low phenotypic ratio for 141 plants was 1:3:1. Quantitative RT-PCR showed a correlation between expression of CYP716A12, CYP72A67, and CYP72A68 genes, all encoding cytochrome P450 enzymes involved in synthesis of hemolytic sapogenins, and the three hemolysis phenotypes. Concurrently, we found that treatment of Caco-2 human colon cancer cells with saponin extracts from four M. truncatula accessions resulted in decreased cell proliferation over time, and that this effect did not appear to be mediated through apoptosis induction. The saponin extracts were analyzed by HPLC-MS to identify individual saponins that could contribute to cytotoxic activity. Therefore, accession differences in M. truncatula saponin accumulation result from differential regulation of saponin synthesis gene expression and inheritance of these differences depends on more than a single gene with dominant and recessive alleles. Saponin extracts are shown to have a negative impact on cancer cell lines.

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