Cloning and sequencing of p- insulin gene isolated from Momordica charantia and in silico analysis of three dimension of protein structure

Diabetes is a disease that the body loses its ability to control blood sugar also it is directly associated with an increased risk of cardiovascular disease. Synthetic hypoglycemic drugs can cause serious side effects. Herbal drugs have potential therapeutic applications because they are effective, have fewer side effects and are of relatively low cost. The Momordica charantia (Linn Family: Cucurbitaceae), whose fruit is known as Karela or bittergourd. Karela is one of the famous vegetables of South Asia, India and is grown in the Amazon, East Africa, Islands in the Caribbean and South America to provide food and medicine. An anti-diabetic agent Polypeptide-P, a 172 amino acid polypeptide isolated from MC seeds. The content of Polypeptide-P in cultivated plants is relatively low and varies widely with tissue, variety, origin and harvest season. Due to impurities such as polysaccharides, the effective ingredient will be less. Gene cloning and expression of this polypeptide in microorganism is a quick and useful alternative for solving such problems. Therefore, in this study the sequence of p-insulin gene from Karela was detected by RT-PCR then sequenced.

RNA was sampled on ripen and unripen fruit from pericarp, seed aril, and seed. Quantification and qualification of extracted RNA was done by spectrophotometer and electrophoreses respectively. Then the RT-PCR reaction was done by p-insulin specific primers and purified by gel electrophoresis and cloned in T-vector plasmid. The recombinant plasmid was extracted and verified to carry p-insulin gene. Then, the recombinant plasmid was done to sequence the p-insulin gene. The 3D structure of p-insulin protein was evaluated by Phyre 2.0 (www.sbg.bio.ic.ac.uk/phyre2/) software.

The electrophoresis of extracted RNA from Karela tissues showed the two 18SrRNA and 28SrRNA bands. A 750 base pair RT-PCR product was amplified by p-insulin specific primers. Also, the RT-PCR result depicted that the p-insulin gene was expressed in aril tissue more than other parts of the fruit. The result showed that the cloning process of p-insulin gene consist of purification of p-insulin PCR production, the ligation reaction and transformation of it’s into the E. coli was successful. Then, the Karela isolated p-insulin gene was sequenced for the first time in Iran. Blasting the plant insulin protein sequence in uniprot database (www.uniprot.org) detected the most important candidate domains including FDA-binding monooxygenase at the N terminal and Prolipoproteindiacyl glyceryltransferase at C terminal of protein.

The low expression of p-insulin and the presence of polysaccharide impurities in Karela reduces the effect of a blood glucose-lowering agent. Therefore, in this study, p-insulin gene of Karela was isolated and sequenced. The sequencing of p-insulin gene will allow its heterologous expression in microorganisms and pure production of p-insulin protein. Key words: Momordica charantia, Diabetes, Cloning, Sequencing, Bioinformatics