Abstract
Plants produce a vast array of metabolites for basic and specialized metabolism. Many of the specialized metabolites are synthesized for defense purposes, to protect the plant from predators, i.e. mammals or insects. One major group of defense compounds is the triterpenoid saponins, which are thought to interact with membrane lipids, thereby disrupting the cell membrane architecture of the predator. Therefore, the use of biotechnologically produced saponins may enable the agricultural industry to fight pests in an environmentally friendly manner.
In the plant Barbarea vulgaris four saponins are identified and found to be responsible for resistance towards flea beetles (Phyllotreta nemorum) and diamondback moths (Plutella xylostella). Genes involved in the biosynthesis of these saponins were recently identified, however the complete biosynthetic pathway is not known to date. These findings set the background of this thesis, which aims to address two main goals: 1) to elucidate additional genes involved in saponin biosynthesis and 2) to study the product specificity for the already identified enzymes.
In order to identify additional genes involved in saponin biosynthesis, a draft genome of B. vulgaris was sequenced and assembled. The draft genome was used to identify UDP-glucose transferase (UGTs) enzymes able to glucosylate the backbone of known saponin structures. These UGTs were found to be organized in tandem repeats and may be the result of gene duplications.
In B. vulgaris P-type and B. vulgaris G-type two 2,3-oxidosqualene cyclases enzymes ,which are the first genes in the pathway leading to saponins, are 98 % identical. Surprisingly, they differ in their product profiles, laying the foundation for the second main part of this thesis. Here, amino acid residues important for the observed product ratios were identified
In vivo results suggest the rate limiting step for saponin biosynthesis in B. vulgaris to be the expression levels of 2,3-oxidosqualene cyclase. To support these findings, an in vivo knock-out of the OSC via CRISPER/Cas was desired. To achieve this, stable transformants of B. vulgaris with the nuclease Cas9 were generated together with a regeneration protocol for B. vulgaris roots and hypocotyls.
In summary, the work described in this thesis led to the identification of genes involved in saponin biosynthesis and the genome organization in B. vulgaris. It further provided a method for the transformation and subsequent regeneration of B. vulgaris. These findings contribute to a better understanding of the fundamentals of plant defense mechanisms which benefit both the scientific community as well as the agricultural sector.
In the plant Barbarea vulgaris four saponins are identified and found to be responsible for resistance towards flea beetles (Phyllotreta nemorum) and diamondback moths (Plutella xylostella). Genes involved in the biosynthesis of these saponins were recently identified, however the complete biosynthetic pathway is not known to date. These findings set the background of this thesis, which aims to address two main goals: 1) to elucidate additional genes involved in saponin biosynthesis and 2) to study the product specificity for the already identified enzymes.
In order to identify additional genes involved in saponin biosynthesis, a draft genome of B. vulgaris was sequenced and assembled. The draft genome was used to identify UDP-glucose transferase (UGTs) enzymes able to glucosylate the backbone of known saponin structures. These UGTs were found to be organized in tandem repeats and may be the result of gene duplications.
In B. vulgaris P-type and B. vulgaris G-type two 2,3-oxidosqualene cyclases enzymes ,which are the first genes in the pathway leading to saponins, are 98 % identical. Surprisingly, they differ in their product profiles, laying the foundation for the second main part of this thesis. Here, amino acid residues important for the observed product ratios were identified
In vivo results suggest the rate limiting step for saponin biosynthesis in B. vulgaris to be the expression levels of 2,3-oxidosqualene cyclase. To support these findings, an in vivo knock-out of the OSC via CRISPER/Cas was desired. To achieve this, stable transformants of B. vulgaris with the nuclease Cas9 were generated together with a regeneration protocol for B. vulgaris roots and hypocotyls.
In summary, the work described in this thesis led to the identification of genes involved in saponin biosynthesis and the genome organization in B. vulgaris. It further provided a method for the transformation and subsequent regeneration of B. vulgaris. These findings contribute to a better understanding of the fundamentals of plant defense mechanisms which benefit both the scientific community as well as the agricultural sector.
| Originalsprog | Engelsk |
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| Forlag | Department of Plant and Environmental Sciences, Faculty of Science, University of Copenhagen |
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| Status | Udgivet - 2017 |