Preliminary In Silico Analysis of Choline Monooxygenase and Betaine Aldehyde Dehydrogenase from Suaeda monoica Forssk. and its Expression in E. coli.

Abstract

Abstract
In a tropical country like India, the yield of crop plants is majorly affected by soil salinity and drought. One of the strategies to address this problem is by introducing genes responsible for salinity/drought tolerance in crop plants. Such genes could be identified and isolated from mangrove plants, as they are known to have evolved certain physiological mechanisms to augment saline environments. One such mechanism is the accumulation of Glycine Betaine, a non-ionic solute that confers osmotic tolerance in most Chenopodiaceae members. Glycine Betaine is known to increase the osmolarity of cells as well as protect the structure of proteins involved in photosynthesis. Its synthesis in plants is brought about by two enzymes, Choline monooxygenase that oxidizes Choline to Betaine aldehyde and Betaine Aldehyde Dehydrogenase that converts Betaine Aldehyde to Glycine Betaine. In this study, an investigation has been made to clone the cDNAs of these two genes originally isolated from a halophyte, Suaeda monoica and study their expression in E. coli by the aid of a protein expression vector, pET32a. PCR amplification with primers designed to amplify the ORF of cDNA clones of SmCMO and SmBADH yielded a product of length 1.3 kb and 1.5 kb respectively. In silico analysis of SmCMO sequence indicates that the ORF of CMO and BADH comprises 1339 and 1508 base pairs respectively. Restriction analysis results suggest that both CMO (Choline Mono-oxygenase) and BADH (Betaine Aldehyde Dehydogenase) ORFs could be cloned in pET32a using BamH1 and Xho1 ends. ClustalW analysis of SmCMO with other reported amino acid sequences indicate the putative consensus sequences of [2Fe-2S] Rieske-type cluster His-168,188 and Cys-166,185 as well as the mononuclear non-heme Fe domain coordinated by Asp-282,286 and His-290,295 essential for CMO activity. Protein sequence analysis using the Protparam tool indicate that CMO is a 50 kDa protein, encoded by 447 amino acids and BADH, a 55kDa protein encoded by 507 amino acids. The theoretical isoelectric point of CMO is 6.05 whereas that of BADH is 5.44.Expression of CMO and BADH in E. coli BL-21(DE3) cells by induction with 1mM IPTG and subsequent SDS-PAGE analysis yielded corresponding molecular weight bands of ~72 KDa and ~77KDa (with thioredoxin fusion protein) respectively. The results indicate that the proteins have been expressed in E. coli and the Salt stress studies show that both CMO as well as BADH transformants were able to grow under 500mM NaCl.

Keywords: ClustalW, Suaeda monoica (Sm), SDS-PAGE, interspecific hybridization, LB (Luria Bertani Broth), CMO, Tetramethylethylenediamine (TEMED), Isopropyl-beta-D-thiogalactopyranoside (IPTG), Ampicillin (Amp), PEG, GSP (Gene specific Primer)

Cite this Article
P. Lakshmi Priya, Kirubanandan
Shanmugam, Hafiz Ansar Rasul Suleria .
Preliminary In Silico analysis of Choline
Monooxygenase and Betaine Aldehyde
Dehydrogenase from Suaeda m onoica
Forssk. and its Expres sion in E. c oli
Research & Reviews: Journal of
Computa tional Biology. 2020; 9(1): 10 3 9 p.