Molecular cloning and functional characterization of a truncated fpsin N-terminus Homology (ENTH) protein from Arabidopsis thaliana

Abstract

Adenylate cyclases (ACs) are enzymes that are known to produce 3ʹ,5ʹ-cyclic adenosine monophosphate (cAMP) from 5ʹ-adenosine triphosphate (ATP) as a result of some associated extracellular stimulations. However, the question of whether or not cAMP does exist in plants has been an issue of debate for a while, mainly due to the less efficient methods employed to isolate this molecule and also because of its very low levels in plants. In contrast to plants, animals and lower eukaryotes ACs and their product cAMP have been firmly established as important signalling molecules with critical roles in cellular signal transduction pathways. Therefore, this study was carried out to determine if higher plants, particularly the Arabidopsis thaliana, have any other additional and functional ACs besides the few and currently known ones and if so, to test for their functional activities. Among the various proteins suspected to harbour an AC catalytic centre in A. thaliana is the epsin N-terminus homology (ENTH), whose truncated form harbouring the AC catalytic center (ENTH-AC) was investigated in this study. In this regard, some A. thaliana plants were initially generated followed by extraction of their total RNA. This total RNA together with some manually designed sequence-specific primers were then used to isolate and amplify the targeted ENTH-AC gene fragment via a specialized RT-PCR system. The isolated ENTH-AC gene fragment was then cloned into a pTrcHis2-TOPO expression vector to yield a pTrcHis2-TOPO:ENTH-AC fusion construct. The produced construct was then used to transform some chemically competent cyaA SP850 mutant E. coli cells, through which its in vivo functional AC activity was then determined and confirmed. After this, more work was then undertaken bioinformatically on the ENTH protein, whereby a wide range of web-based softwares and computer-based programs were used to determine the exact functional role(s) of this protein in Arabidopsis and possibly, other related higher plants. All in all, findings from this study unequivocally indicated that the ENTH protein is a bona fide functional higher plant AC with a central role in cAMP-mediated fungal stress responses.