Isolation of cDNAS of genes involved in programmed cell death and construction of an expressed sequence tag database for Aponogeton madagascariensis

The lace plant, Aponogeton madagascariensis is an aquatic plant that forms perforations in its leaves as a part of its normal developmental growth. This process of perforation formation is extremely regulated and has been shown to be orchestrated by developmental programmed cell death (PCD). The accessibility and predictability of perforation formation, and the propagation of lace plant in sterile conditions, make this an attractive model system for the study of developmentally regulated PCD in plants; however, to date no molecular work has been carried out in this species. In this study, protocols for RNA extraction, cDNA synthesis and isolation of cDNA for target genes were optimised. Also, cDNAs for some of the genes associated with PCD were isolated. Through the degenerate primers approach a 288 bp fragment of an ethylene receptor (ETR1) cDNA was isolated. This fragment is part of an ETR1 functional domain, sensor domain. A 1002 bp fragment of lace plant polyubiquitin 10 homologue was also isolated. This fragment is comprised of a 681 bp protein coding region and a 321 bp untranslated region. The mRNA levels of both ETR1 and ubiquitin at the different stages of perforation formation were also investigated. Ubiquitin showed equal mRNA levels across all the different stages of lace plant perforation formation while ETR1 mRNA levels were higher in window stage leaves as compared to pre-perforation and mature stage leaves. Based on these results, a model for ETR1 expression during lace plant perforation formation was proposed. Preliminary experiments in isolation of lace plant metacaspases were also carried out. Some metacaspase primers amplified different size fragments in lace plant but these fragments were not sequenced. An EST database for window stage leaves was also constructed. This database consists of a total of 147 clones, 97 of which are unique. Some of the clones, such as the 20S proteasome beta-subunit, cytochrome P450 monooxygenase and WRKY transcription factors are cDNAs for genes that playa role in PCD in other plant species.