Molecular Cloning and Functional Analysis of LEAFY COTYLEDON2 Gene of Theobroma cacao.
Open Access
- Author:
- Clemens, Adam Winfield
- Area of Honors:
- Biotechnology
- Degree:
- Bachelor of Science
- Document Type:
- Thesis
- Thesis Supervisors:
- Mark Guiltinan, Thesis Supervisor
David Scott Gilmour, Thesis Supervisor
Donald Ashley Bryant, Thesis Honors Advisor
Scott Brian Selleck, Faculty Reader - Keywords:
- Molecular Cloning
Functional Analysis
LEAFY COTYLEDON2
Theobroma cacao
fatty acids
somatic embryogenesis - Abstract:
- Arabidopsis thaliana LEAFY COTYLEDON2 (AtLEC2) is a transcription factor that acts to regulate transcription of genes during embryogenesis through its B3 domain and affect seed development in a complex, yet only partially understood manner, including the induction of morphogenic events and the synthesis of seed storage proteins and metabolites. In the model plant Arabidopsis thaliana, AtLEC2 has been shown to regulate genes within both the morphogenesis and maturation phases of embryo development. Using the sequence of LEC2 from Arabidopsis and the sequenced genome of cacao, a LEC2 candidate homolog from Theobroma cacao, the Chocolate Tree, (TcLEC2) was cloned by PCR using cDNA transcribed from RNA extracted from Scavina6 cacao tissue. The purpose of this study was to obtain evidence for the function of the candidate LEC2 gene in cacao, and to determine its potential role in the synthesis of fatty acids, key components of the cacao seed. Using Agrobacterium tumefaciens-mediated transformation, TcLEC2 was transiently expressed in cacao Scavina6 genotype leaves and the synthesis of fatty acids was determined. This transient assay method was developed and optimized as part of this study. The results of TcLEC2 expression on leaf fatty acid biosynthesis are inconclusive and further testing is in progress. It is possible that the TcLEC2 candidate gene is not capable of inducing fatty acid biosynthesis in seeds, possibly because some other required factors are not present. The second aspect of this study was to determine the regulatory effects of TcLEC2 on induction of somatic embryogenesis, a process similar to zygotic embryogenesis but initiated in vitro with the use of plant hormones. TcLEC2 was expressed in cacao primary somatic cotyledons and petal tissue using A. tumefaciens-mediated transformation. Expression of TcLEC2 was found to significantly increase the cacao stable transformation efficiency in somatic tissue. TcLEC2 was also shown to lead to the development of large clusters of embryonic structures on both callus of explants and transgenic somatic embryo cotyledons. This abnormal development of clustered embryonic structures indicates that the over-expression of TcLEC2 induces the embryogenesis pathway. This result supports the conclusion that TcLEC2 is a functional ortholog of the Arabidopsis LEC2 gene and functions in the control of embryogenesis.