Design of Antisense against Blp Gene and Investigation of the Gene Knock down Effect on the Bacteriorhodopsin Production in the Halobacterium Salinarum

Abstract

Background: Bacteriorhodopsin is a transmembrane protein has been found in the membrane of Halobacterium salinarum as a light dependent proton pump. This protein is a type of photochromic protein and many studies has been done about it. Because of its important functions in researches, optimum production of this protein is essen- tial. In spite of blp role in the production of bacteriorhodopsin, its function has remained unknown yet. However, it is possibility acts as inducer of bacteriopsin which in turn is precursor of bacteriorhodopsin. Finding the role of this protein as inducer of bacterioopsin make the production of bacteriopsin more clear. The main purpose of this study is to identify the role of blp in bacteriorhodopsin network production through antisense gene experiments. In order to achieve this purpose, the antisense sequence was designed against blp. Then its effect on the bacteri- orhodopsin production investigated. Materials and Methods: The researchers designed primers for a 180 bp area that contains ribosome binding site and initial part of the blp. Then restriction sites of BamH1 and Xba1 enzymes inserted at the 5’ end of them with 5 random nucleotides at the end of each primers. The primers analyzed by oligoanalyzer software. the fragment amplified by PCR. It was cloned in to pBluscript SKII cloning vector. In order to add the sequences of promoter and terminator the fragment was cloned between these elements on the pVDHS1 and re-amplified along with terminator and promoter.In the next step, the fragment was cloned into pA- MAX and vector was transformed to the Halobacterium salinarum after transformation and validating by PCR and enzymatic digestion of vector, bacterium placed under circumstances of induction for induction the expression of antisense. in order to induce the expression of antisense the promoter activate at the concentration of 0.3 Mm of KCl and and deactivate at 100mM KCl. After antisense expression, the function of antisense in bacterium investi- gated trough RTPCR technique Then the amount of bacteriorhodopsin in both induced and uninduced culture. Results: finally،the researchers found that silencing of blp gene through its specific anti-sense molecules led to decrease of bacteriorhodopsin production .its more blp gene has been decrease of bacteriorhodopsin production and it is more likely that this gene acts as an inducer in bacteriorhodopsin network production.