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

Document Type : Research Paper

Authors

1 Assistant Professor, Department of Agricultural Engineering, Payame Noor University,

2 MSc Student, Department of Cell and Molecular Biology, Faculty of Biological Sciences, Kharazmi University, Tehran, Iran

Abstract

Objective
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.
 
Materials and methods
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).
Results
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.
 
Conclusions
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.

Keywords

References

عرب پور رق آبادی زهرا، محمدآبادی محمدرضا، خضری امین (1400) الگوی بیانی ژن p32 در بافت‌های ران، دست، راسته و چربی پشت بره کرمانی. مجله بیوتکنولوژی کشاورزی، 13(4)، 183-200.
محمدآبادی محمدرضا (1399) بیان ژن ESR1 در بز کرکی راینی با استفاده از real time PCR‎. مجله بیوتکنولوژی کشاورزی 12(1)، 192-177.
محمدآبادی محمدرضا (1399) پروفایل بیانیmRNA مختص بافت ژن ESR2 در بز. مجله بیوتکنولوژی کشاورزی 12(4)، 184-169.
محمدآبادی محمدرضا، سفلایی محمد (1399). پروفایل بیانی mRNA مختص بافت ژن BMP15 در بز. مجله بیوتکنولوژی کشاورزی 12(3)، 208-191.
محمدآبادی محمدرضا، کرد محبوبه، نظری محمود (1397) مطالعه بیان ژن لپتین در بافت‌های مختلف گوسفند کرمانی با استفاده از real time PCR. مجله بیوتکنولوژی کشاورزی 10(3)، 122-111.
References
Apak R, Calokerinos A, Gorinstein S, et al. (2022) Methods to evaluate the scavenging activity of antioxidants toward reactive oxygen and nitrogen species (IUPAC Technical Report). Pure Appl Chem 94(1), 87-144.
Ara A, Ahmed KA, Xiang, J (2018). Mannose-6-phosphate receptor: a novel regulator of T cell immunity. Cell Mol Immunol 15(11), 986-988.
Bhauso TD, Radhakrishnan T, Kumar A, Mishra GP, et al. (2014) Overexpression of bacterial mtlD gene in peanut improves drought tolerance through accumulation of mannitol. Sci World J 19, 2014
Beiranvand S, Piri‐GharaghieT, Dehganzad B, et al. (2022) Novel NAD‐independent Avibacterium paragallinarum: Isolation, characterization and molecular identification in Iran. Vete Med Sci 8(3), 1157-1165.
Callender P, Wettig D (2021) Phase Behavior of Non‐Ionic Surfactant‐Medium Chain Triglyceride‐Water Microemulsion Systems. J Surfactants Deterg 24(4), 603-629.
Ghajari G, Nabiuni, M, Amini E (2021) The association between testicular toxicity induced by Li2Co3 and protective effect of Ganoderma lucidum: Alteration of Bax & c-Kit genes expression. Tissue Cell 72, 101552.
Fita A, Rodríguez-Burruezo A, Boscaiu M, et al. (2015) Breeding and domesticating crops adapted to drought and salinity: a new paradigm for increasing food production. Front Plant Sci 6, 978.
Hahn G, Kaup B, Bringer-Meyer S, Sahm H (2003) A zinc-containing mannitol-2-dehydrogenase from Leuconostoc pseudomesenteroides ATCC 12291: purification of the enzyme and cloning of the gene. Arch Microbiol 179(2), 101-107.
Jannapureddy S, Sharma M, Yepuri G, et al, (2021) Aldose reductase: an emerging target for development of interventions for diabetic cardiovascular complications. Front Endocrinol 12, 636267.
Kanayama Y, Yamaki S (1993) Purification and properties of NADP-dependent sorbitol-6-phosphate dehydrogenase from apple seedlings. Plant Cell Physiol 34(6), 819-823.
Khalil SR, Ibrahim AS, Hussien BA, et al. (2017) Cloning of a functional mannose-6-phosphate reductase (M6PR) gene homolog from Egyptian celery plants (Apium graveolens): overexpression in non-mannitol producing plants resulted in mannitol accumulation in transgenic individuals. 3 Biotech 7(5), 1-12.
Ladhari A, Zarrelli A, Di Meo MC, et al. (2022) Physiological mechanisms and adaptation strategies of Lactuca sativa L. in response to Olea europaea L. and Ficus carica L. allelochemicals. S Africa J Botany 147, 106-118.
Moenga SM, Gai Y, Carrasquilla‐Garcia et al. (2020) Gene co‐expression analysis reveals transcriptome divergence between wild and cultivated chickpea under drought stress. Plant J 104(5), 1195-1214.
McParland EL, Alexander H, Johnson WM (2021) The osmolyte ties that bind: genomic insights into synthesis and breakdown of organic osmolytes in marine microbes. Front Mar Sci 8, e2021.
Meena M, Prasad V, Zehra A, et al. (2015) Mannitol metabolism during pathogenic fungal–host interactions under stressed conditions. Front Microbiol 6, 1019.
Msomi NZ, Erukainure OL, Islam MS (2021) Suitability of sugar alcohols as antidiabetic supplements: A review. J Food Drug Anal 29(1), e1.
Piri‐Gharaghie T, Beiranvand S, Riahi A, et al. (2022) Fabrication and characterization of thymol‐loaded chitosan nanogels: Improved antibacterial and anti‐biofilm activities with negligible cytotoxicity. Chem Biodivers 19(3), e202100426.
Piri-Gharaghie T, Jegargoshe-Shirin N, Saremi-Nouri S, et al. (2022) Effects of Imipenem-containing Niosome nanoparticles against high prevalence methicillin-resistant Staphylococcus Epidermidis biofilm formed. Sci Rep 12(1), 1-13.
Piri-Gharaghie T, Doosti A, Mirzaei SA (2022) Fabrication and Characterization of pcDNA3. 1 (+) Location within Chitosan/Nanoparticles Complexes for Enhanced Gene Delivery. Iran J Biotechnol 20(3), 88-100.
Piri-Gharaghie T, Doosti A, Mirzaei SA (2022) Identification of Antigenic Properties of Acinetobacter baumannii Proteins as Novel Putative Vaccine Candidates Using Reverse Vaccinology Approach. Appl Biochem Biotechnol 7, 1-23.
Pleyerová I, Hamet J, Konrádová H, et al. (2022) Versatile roles of sorbitol in higher plants: luxury resource, effective defender or something else. Planta 256(1), 1-27.
Sánchez J, Harwood JL (2002) Biosynthesis of triacylglycerols and volatiles in olives. Europ J Lipid Sci Technol 104(9‐10), 564-573.
Slavin J (2003) Impact of the proposed definition of dietary fiber on nutrient databases. J Food Compos Anal 16(3), 287-291.
Williamson JD, Stoop JM, Massel MO, et al. (1995) Sequence analysis of a mannitol dehydrogenase cDNA from plants reveals a function for the pathogenesis-related protein ELI3. Nat Acad Sci 92(16), 7148-7152.
Walker RP, Battistelli A, Bonghi C, et al. (2020) Non-structural carbohydrate metabolism in the flesh of stone fruits of the genus Prunus (Rosaceae)–A review. Fron Plant Sci 11, 549921.
Xiaotang DI, Van Den Burg H, Takken FLW (2018) Plants comprising pathogen effector constructs. Universiteit Van Amsterdam 7, e2018.
Yang J, Zhu L, Cui W, et al. (2018) Increased activity of MdFRK2, a high-affinity fructokinase, leads to upregulation of sorbitol metabolism and downregulation of sucrose metabolism in apple leaves. Hort Culture Res 5, e2018.
Zwiewka M, Nodzyński T, Robert S, et al. (2015) Osmotic stress modulates the balance between exocytosis and clathrin-mediated endocytosis in Arabidopsis thaliana. Mol plant 8(8), 1175-1187.
Agricultural Biotechnology Journal
Volume 15, Issue 2
June 2023
Pages 199-216

Files

Share

How to cite

Statistics