In collaboration Iranian Medicinal Plants Society

Document Type : Research Paper

Author

Associate Professor of Plant Genetics engineering, Plant Science and Medicinal Plants Branch, Meshgin Shahr Faculty of Agriculture, University of Mohaghegh Ardabili, Ardabil, Iran

Abstract

The existence of isoform genes in plants led to the creation of the different types of isomeric metabolites like these glycosylated forms. This study was conducted to investigate the identification and heterologous expression of Saffron stigmas Glycosyl transferase coding isoform gene during the pollination stage. The results have been obtained by sequencing and bioinformatics analysis of an isolated gene from saffron genomes by degenerate oligo’s revealed that the gene is in1283 bp length and belongs to CsUGT protein family which has Apo- plastic secretion in the cell. to evaluate the enzymatic function, firstly the isolated sequence was sub-cloned under arabinose induce promoter in pThio-UGT expression vector by Gibson assembly technique, then the recombinant vector transformed into BL21-pGro7 bacteria which were able to express chaperon proteins with EL& ES subunits. Followed by destructing the bacteria cell wall via ultrasound, the pellet was suspended by PBS solution and then the soluble proteins were extracted by boiling method. Finally, the protein electrophoresis by SDS pages10% was showed that the recombinant protein of CsUGT expressed correctly in bacteria with 69/5 kDa molecular weight. The gained results in this project could be applied to determine the breeding's strategies to improve qualitative and quantitative traits such as color and aroma in saffron.

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Gibson, D.G., Young, L., Chuang, R.Y., Venter, J.C., Hutchison, C.A., and Smith, H.O. 2009. Enzymatic assembly of DNA molecules up to several hundred kilobases. Nature Methods 6 (5): 901-903.
Gosch, C., Halbwirth, H., Schneider, B.,
 
Hölscher, D., Stich, K. 2010. Cloning and heterologous expression of glycosyltransferases from Malus x domestica and Pyrus communis, which convert phloretin to phloretin 2′-O-glucoside (phloridzin). Plant Science 178 (3): 299- 306.
Hansen, K.S., Kristensen, C., Tattersall, D.B., Jones, P.R., Olsen, C.E., Bak, S., and Møller, B.L.  2003. The in vitro substrate regiospecificity of recombinant UGT85B1, the cyanohydrin glucosyl transferase from Sorghum bicolor. Phytochemistry 64 (1): 143-151.
Hosseinpour, A.N., Nematzadeh, G.A., Giuliano G., Ranjbar G.A., and Yamchi, A. 2017.  Identification of Apo- Carotenoids' crocin and crocetin isomers in saffron crude extracts by HPLC coupled to atmospheric pressure chemical ionization and high resolution orbitrap mass spectrometry. Saffron Agronomy and Technology 4 (4): 291-300.
Kumar, R., Sangwan, R.S., Mishra, S., Sabir, F., and Sangwan, N.S. 2012. In silico motif diversity analysis of the glycon preferentiality of plant secondary metablic glycosyltransferases. Plant Omics 5 (3): 200-210.
Li, Y., Luo, H.M., Sun, C., Song, J.Y., Sun, Y.Z., Wu, Q., Wang, N., Yao, H., Steinmetz, A., and Chen, S.L. 2010. EST analysis reveals putative genes involved in glycyrrhizin biosynthesis. BMC genomics 11 (1): 268.
Lunkenbein, S., Bellido, M., Aharoni, A., Salentijn, E.M., Kaldenhoff, R., Coiner, HA., Munoz Blanco, J., and Schwab, W. 2006. Cinnamate metabolism in ripening fruit. Characterization of a UDP-glucose: cinnamate glucosyltransferase from strawberry. Plant physiology 140 (3): 1047-1058.
Demurtas, O. C., Frusciante, S., Ferrante, P., Diretto, G., Hosseinpour Azad, N., Pietrella, M., and Al Babili, S. 2018. Candidate enzymes for saffron crocin biosynthesis are localized in multiple cellular compartments. Plant physiology 177 (3): 990-1006.
Moraga, A.R., Nohales, P.F., Perez, J.A., and Gomez-Gomez, L. 2004. Glucosylation of the saffron apocarotenoid crocetin by a glucosyltransferase isolated from Crocus sativus stigmas. Planta 219 (6): 955-966.
Ono, E., Fukuchi-Mizutani, M., Nakamura, N., Fukui, Y., Yonekura-Sakakibara, K., Yamaguchi, M., Nakayama, T., Tanaka, T., Kusumi, T., and Tanaka, Y. 2006. Yellow flowers generated by expression of the aurone biosynthetic pathway. Proceedings of the National Academy of Sciences of the United States of America 103 (29): 11075–11080.
Ono, E., Ruike, M., Iwashita, T., Nomoto, K., and Fukui, Y. 2010. Co-pigmentation and flavonoid glycosyltransferases in blue Veronica persica flowers. Phytochemistry 71 (7): 726-735.
Subramanian, S., Stacey, G., and Yu, O. 2007. Distinct crucial roles of flavonoids during legume nodulation. Trends Plant Science 12 (7): 282-285.
Wang, J., and Hou, B. 2009. Glycosyltransferases key players involved in the modification of plant secondary metabolites. Front Biol China 4 (1): 36–46.
Woo, H.H., Jeong, B.R., Hirsch, A.M., and Hawes, M.C. 2007. Characterization of Arabidopsis AtUGT85A and AtGUS gene families and their expression in rapidly dividing tissues. Genomics 90 (1): 143-153.
Yonekura-Sakakibara, K., and Hanada, K. 2011. An evolutionary view of functional diversity in family 1 glycosyltransferases.