TRANSIENT TRANSFORMATION OF THE CYP7A1 GENE FOR CHOLESTEROL CONVERSION IN TOBACCO PLANTS (NICOTIANA TABACUM) AND REVIEW OF URSODEOXYCHOLIC ACID

• Main Author: Megalestin Raunsai, Marlin
• Format: Theses
• Language: Indonesia
• Online Access: https://digilib.itb.ac.id/gdl/view/51682
• Institution: Institut Teknologi Bandung

Ursodeoxycholic acid (UDCA) is a secondary bile acid derived from cholesterol in hepatocytes. UDCA is used for the treatment of Primary Billier Cirrhosis (PBC) and has generally been applied for the treatment of various liver diseases. The cytochrome P450 7A1 gene (CYP7A1) encodes the cholesterol 7?-hydroxylase enzyme which plays a role in the initial step of the UDCA biosynthesis from cholesterol through the classic pathway. The insertion of the CYP7A1 gene into plants containing cholesterol can be mediated by Agrobacterium tumefaciens. This study aims to transform the CYP7A1 gene into A. tumefaciens and to analyze the conversion result of this gene using the transient transformation technique on Nicotiana tabacum plants. Electrocompetent cells were prepared using A. tumefaciens strain LBA4404. The CYP7A1 gene used in this study was isolated from chickens from the previous studies. The CYP7A1 gene was cloned into the pGEM- T EASY vector and was confirmed by sequencing and then cloned into the pCAMBIA1303 expression vector using restriction enzymes, BglII and SpeI. Transformation of pCAMBIA1303 and pCAMBIA-CYP7A1 plasmids to A. tumefaciens by electroporation used Xcell Gene Pulser. Confirmation of the transformation results was analyzed by PCR using CaMV35S and GUS primers. The seeds of N. tabacum cultivar SR1 were grown on solid MS0 media. The transient transformation used A. tumefaciens containing pCAMBIA1303 and pCAMBIA-CYP7A1 on MS medium added with acetosyringone and silwet S-408. This transformation used leaf explants from the apical meristem of N. tabacum. Confirmation of the transient transformation was tested by the histochemical GUS assay. The results of the plasmid transformation to A. tumefaciens LBA4404 containing pCAMBIA1303 and pCAMBIA1303-CYP7A1 were successful in the presence of colonies growing on the selection medium and in the results of electrophoresis which produced band sizes of about 641 bp and 2100 bp, respectively. The N. tabacum cultures were successful in subculture and the age of tobacco plants used for transient transformation was 8 weeks old. Histochemical GUS assay has been tested once but has not been successful because no blue spots were found in tobacco explants. Conclusion: pCAMBIA1303 and pCAMBIA CYP7A1 plasmids were successfully transformed to A. tumefaciens LBA4404 but A. tumefaciens LBA4404 contains pCAMBIA1303 and pCAMBIA1303-cyp7A1 have not been successfully transformed to N. tabacum which indicates plasmid or the gene has not been successfully inserted. The research was continued but was constrained by the closure of laboratory access due to the Covid-19 outbreak so that a literature review was carried out to complement the research data. In the review article, 56 literatures were collected and analyzed.