Genes implicated in phorbol ester (PE) biosynthesis and interception through downregulation of casbene synthase genes

• Main Author: Ng, Ailing
• Other Authors: Hong Yan
• Format: Theses and Dissertations
• Language: English
• Published: 2014
• Subjects: DRNTU::Science::Biological sciences::Molecular biology; DRNTU::Science::Biological sciences::Biochemistry
• Online Access: http://hdl.handle.net/10356/61541
• Institution: Nanyang Technological University

Phorbol Esters (PE) are naturally occurring tigliane diterpenoids accumulating mostly in plants of Euphorbiaceae family. They were found to cause undesirable biological effect in mammals including inflammation, tumour promotion, cell division and proliferation. PE are found in all plant parts of Jatropha curcas L. (JC or Jatropha), a multipurpose oil bearing shrub belonging to the family of Euphorbiaceae. The presence of PEs in Jatropha especially their seed oil and cake rendered them unsuitable for human and animal consumption. This proposed research aims to clone and characterize genes critical for PE biosynthesis, to set up necessary platform technology, and to explore the possibility to reduce PE levels in Jatropha. First, different Jatropha accessions collected from different regions / countries, including China, India, South America, Indonesia and Africa were screened. PE level in Jatropha stem, root, leaf, flower and seed was investigated. Jatropha seed was found to contain the highest amount of PE in seed's inner skin. Subsequently, genes that are possibly involved in PE biosynthesis were identified by bioinformatics approaches. A boutique array consisting of 112 candidate genes was constructed using an in house microarray facility. Expression level of these genes in subjects that have high PE content was compared against embryo and cotyledon that has non-detectable PE level. There were 67 genes that were upregulated in inner skin and 22 genes that were also upregulated in Croton tiglium (Croton) seed. Both Jatropha inner skin and Croton seed have high PE content. Twelve genes that were upregulated in both inner skin and Croton seed were identified. They include two homologs to terpene synthase genes and ten homologs to modification enzymes. Preferential expression of these genes in inner skin was confirmed by quantitative real time PCR. Virus Induced Gene Silencing (VIGS) using tobacco rattle virus which induces RNA interference (RNAi) silencing transiently in Jatropha leaves was used to characterize selected candidate genes' functions in PE synthesis. The candidates include Jatropha homologs to geranylgeranyl diphosphate (GGPP) synthase, terpene synthases and casbene synthases (a diterpene synthase in castor bean). Our preliminary finding demonstrated that single knockdown of GGPP synthase, terpene synthase and casbene synthases ensued a reduction of up to 80% of PE content in the leaves of Jatropha. A double knockdown of two candidate genes reduced PE content in the leaves to less than 15% of control. Later, function of promising gene candidates was investigated by stable transformation system with RNAi knockdown approach. Transformation of Jatropha via two regeneration platform, somatic embryogenesis and organogenesis was employed. Molecular characterization and integration pattern of transgene was investigated on T0 transgenic Jatropha lines. Casbene synthase genes isolated from Jatropha genome were found to be promising candidates. VIGS has shown that transient gene suppression of casbene synthase genes led to phorbol ester reduction in leaves. In vitro functional analysis proved the activity of CasA 163 of converting GGPP into casbene. Genetic modification with a seed specific promoter driving RNAi construct targeting function domains of CasA 163 gene was proven effective as shown in their transcript level reduction. More importantly, efficient CasA 163 gene suppression has led to downregulation of PE level in Jatropha seeds. In summary, this thesis work has led to the identification of several genes that are potentially involved in various steps of phorbol ester biosynthesis. Significantly, a casbene synthase homolog’s enzymatic activity of converting GGPP into casbene was confirmed by an in vitro system. Moreover, stable transformation with an RNAi construct driven by a seed specific promoter was successful in reducing its transcript level as well as phorbol ester level. All data consistently point to its critical function in biosynthesis of casbene, which is a critical precursor to phorbol esters. GM Jatropha with seed specific suppression of CasA 163 or other PE biosynthetic genes could possibly address the concern on Jatropha seed and oil toxicity. Effective reduction of phorbol ester in Jatropha helps address environmental concerns on large scale plantation of Jatropha, making it less hazardous for processing Jatropha oil and biodiesel and more importantly, raise the value of seed cake by making it a possible animal feed.