Molecular identification and functional characterization of a novel adenylyl cyclase from Glycine max

Abstract

The overall aim of this research was to identify and characterise a predicted adenylyl cyclase (AC) enzyme in Glycine max; accession number XP_003529590; gene ID Glyma.07G251000. To start with, a preliminary bioinformatic analysis of the XP_003529590 gene was performed prior to the practical experimental work so as to gain a better understanding of the gene annotation, gene expression profile and its secondary structure. After that, total mRNA was then isolated from the soybean plant followed by amplification of the targeted XP_003529590 gene via RT-PCR and its subsequent cloning into the pTRcHis2-TOPO TA cloning vector. The successfully cloned XP_003529590 was then used to transform some chemically competent E. coli BL21 (DE3) pLysS expression cells followed by recombinant protein expression through induction with 1 mM of isopropyl-β-D-thiogalactopyranoside (IPTG). The expressed recombinant protein was herein referred to as GmAC1. After the expression, the ability of the expressed recombinant GmAC1 protein to generate cyclic adenosine monophosphate (cAMP) within the transformed cells was then assessed and determined endogenously using the enzyme immunoassaying system. An establishment of the actual AC activity of the recombinant GmAC1 protein was then undertaken via a complementation system using the SP850 E. coli mutant strain. After confirmation of the AC activity, expression of the GmAC1 protein was upscaled, followed by its affinity purification on a HisPur Ni-NTA resin matrix. After purification, an in vitro characterisation of the GmAC1's enzymatic activity was then undertaken using the enzyme immunoassaying system. Finally, the probable physiological roles of the XP_003529590 gene in soybean were then assessed and established through bioinformatic analysis. Consequently, the undertaken preliminary bioinformatic analysis showed that the gene ID for the XP_003529590 is Glyma.07G251000 (Glyma_07G251000), which is primarily expressed during the primary root development and in the primary meristems, and its protein product being a nucleic acid and/or compound binding alpha-helical pentatricopeptide protein. In addition, the undertaken endogenous assaying of the expressed recombinant GmAC1 protein showed that this protein could enhance cAMP production in the transformed bacterial cells to about ≥ 3.0 folds. Eventually, the complementation testing then practically confirmed that the expressed recombinant GmAC1 protein is indeed a bona fide AC molecule as it could physiologically rescue the mutant SP850 E. coli host from being a non-lactose fermenter to a lactose fermenter. Subsequently, the in vitro characterisation of the GmAC1 showed that the recombinant protein was indeed a soluble AC (sAC) as its activity could be positively enhanced by the Mn²⁺, Ca²⁺, HCO₃⁻ molecular ions and not the F- ion. Finally, the physiological evaluation of the XP_003529590 through bioinformatics strongly predicted its primary role in abiotic and biotic stress tolerence particularly during the juvenile developmental stages of the soybean plant. Therefore, the researched XP_003529590 or GmAC1 protein can be a very useful molecular component in possible further research to produce transgenic plants/crops that are tolerant to abiotic stresses such as drought, cold, flooding and salinity that affect crop plants during their early developmental stages.