Cloning and investigation of celery plant Mannose-6-phosphate reductase (M6PR) gene expression in E. coli bacteria to control the catabolism pathway

Compared to other primary photosynthetic products (such as sucrose and starch), little is known about sugar metabolism and how it is integrated with others. Mannose-6-phosphate reductase (M6PR) is a key enzyme involved in mannitol biosynthesis in celery. This study aimed to clone the gene, express and purify the M6PR enzyme and investigate its function on mutated genes in a laboratory environment.
 
First, the mRNA was extracted from the celery plant, then the cDNA was synthesized, and the product was used as a template to amplify the M6PR gene. The PCR product was purified in a DNA gel extraction kit. The purified PCR product was cloned into the pTZ57R vector according to the T/A Cloning recipe (Fermentase Company). Susceptible cells of E.coli strain Top10 were prepared using the biochemical method of calcium chloride, and the recombinant vector inside it was transformed and cultured on a plate containing ampicillin. The cloning accuracy was done using M6PR gene PCR and enzymatic digestion of the recombinant plasmid by BamHI and SacI enzymes (Fermentase Company). The M6PR gene was homogenized in the expression plasmid pET32a and transferred into E.coli strain BL2I. The promoter was induced with IPTG and analyzed by western blotting. The protein was purified by affinity chromatography column (His. Tag/S.Tag).
The results showed that the enzyme could identify the heteroduplex regions of the gene. The recombinant M6PR purified from Escherichia coli had specific molecular activity. The results of double digestion of the plasmid with SacI and BamHI enzymes were 2870 bp and 186 bp fragments. According to the blast test result, the current fragment had 100% similarity with the M6PR gene of the celery plant. M6PR recombinant gene transcription results showed that the M6PR recombinant gene transcription rate was 2.3 in the transgenic strains and 0.32 in control, which showed a statistically significant difference at the P<0.01 level. After induction of the promoter and sampling at different times, the samples on the SDS-PAGE gel showed a protein band in the region of 42 kDa, indicating the protein's successful expression.
 
Homology of M6PR enzyme gene obtained from celery plant and then recombinant production of this enzyme in the laboratory can lead to its high expression in the prokaryotic system so that the enzyme has activity. Also, the present study showed that plant enzymes are active when expressed in bacteria and can be used as a suitable source to accelerate catabolic activity.