Specific expression of exendin-4 gene in carrot root (Daucus carota L.) under regulation of MLL promoter

Exendin-4 (Ex4) is a 39-amino acid peptide isolated from the salivary secretions of the lizard Heloderma susceptum and it has similar biological function as glucan-like peptide (GLP-1) receptor. The most important physiological role of GLP-1 is to regulate the amount of glucose metabolism by reducing the entry of glucose into the cells. Transgenic plants can be used as factories for the production of recombinant proteins. In order to maximize the efficiency of transgenic plants, the expression of foreign genes can be regulated in terms of time and place under the regulation of different promoters. Considering the nutritional value of carrot tuber and its fresh consumption, in this study, CTBEx4 gene was expressed in carrot plant using a root-specific promoter. In this study, in order to specifically express the CTBEx4 gene in carrot root, the Major Latex-Like promoter (MLL) related to the specific expression in sugar beet storage root tissue was replaced by the CaMV35S promoter in the pBI121 construct and were used for the transformation of carrot explants.

Using the CTAB method, DNA was extracted from the leaves of the sugar beet plant, then the PCR reaction was performed using specific MLL primers. The obtained fragment was replaced by the CaMV35S promoter in the pBI121 construct using restriction enzymes. Both pBI121-Mll-CTBEx4 and pBI121-CamV35S-CTBEx4 constructs were used to transform carrot leaf explants using Agrobacterium tumefaciens bacteria. Transgenic explants were indirectly regenerated into plants in MS culture media in vitro and then transferred to pot. Finally, the CTBEx4 gene expression of the transgenic plants was analyzed at mRNA and protein levels using RT-PCR and ELISA.

The results of the expression analysis using RT-PCR and ELISA methods indicated that the expression of CTBEx4 gene in carrot root tissue was regulated by MLL promoter, while no expression was observed in leaf tissue. Also, the results showed that the CTBEx4 gene was expressed under CaMV35S promoter regulation in both root and leaf tissues, but its expression in roots was lower (30%) than that under MLL promoter regulation. The obtained results indicated the ability of the MLL promoter to specific expression the CTBEx4 gene in the roots of carrot plants.

The MLL promoter was able to express the CTBEx4 gene in the root tissue of carrot plants at the mRNA and protein level, Also, CTBEx4 protein under MLL promoter had higher expression level compared to CaMV35S promoter.