Datensatz

Zusammenfassung

Recently a BLAST search of the Arabidopsis genome using a 14-mer motif with specificity for ATP binding and catalysis resulted in the identification an AC-like protein coded for by the At3g21465 gene. However, the AC-like protein has not yet been shown to possess any known putative AC catalytic function but instead, it only appears to be transcriptionally up-regulated in response to biotic stress factors. Therefore in an attempt to test and determine whether this putative protein candidate has any functional AC activity, total mRNA of the 4-6 weeks old Arabidopsis thaliana plants was extracted and used as a template for the complementary synthesis and amplification of a 384 bp AC-like gene fragment via a specialized Reverse Transcriptase - (RT-PCR) system. The amplified fragment was cloned into a pTrcHis2-TOPO expression vector and the resultant recombinant expression vector transformed into chemically competent E. cloni EXPRESS BL21 (DE3) pLysS expression host cells. Positive clones were determined by PCR and further validated by nucleotide- specific sequencing. The 18.0 kDa C-terminus His-tagged recombinant AC-like protein was then over- expressed following an induction with isopropyl-β-D-1-thiogalactopyranoside (1 mM, IPTG) and purified over a Ni-NTA affinity matrix system. The endogenous and in vitro AC activities of the resultant recombinant AC-like protein were then tested via a cAMP-linked enzyme immunoassaying system while its inherent in vivo AC activity was also concurrently tested via a complementation testing system using the cyaA SP850 mutant Escherichia coli cells. Results from these three independent assays collectively indicated that the AC-like protein encoded for by the At3g21465 gene from A. thaliana possesses the endogenous, in vitro and in vivo AC activities, and thus confirming it as a higher plant AC molecule with a possible cAMP-mediated signaling system.