PENGARUH CEKAMAN OKSIDATIF FE2+ TERHADAP PERTUMBUHAN DAN EKSPRESI GEN-GEN TERKAIT BIOSINTESIS ASTAKSANTIN DALAM KULTUR SPIROGYRA SP

• Main Author: Idzaturrohim, Salimah
• Format: Final Project
• Language: Indonesia
• Online Access: https://digilib.itb.ac.id/gdl/view/63278
• Institution: Institut Teknologi Bandung

Fe2+ oxidative stress in the culture of Spirogyra sp. is thought to affect growth and can increase the accumulation of astaxanthin, a metabolite derived from carotene which has antioxidant properties. Astaxanthin biosynthesis is influenced by several genes related to biosynthetic pathway of astaxanthin including BKT (?-carotene ketolase) and CrtR-B (?-carotene hydroxylase) genes. This study was aimed to analyze the effect of Fe2+ stress on the growth and expression of the astaxanthin biosynthesis-related genes in the culture of Spirogyra sp. Samples of Spirogyra sp. were collected from the fishing pond in Padalarang, West Bandung. Six gram of fresh weight of Spirogyra sp. filamen was inoculated and cultivated in 2 l of 2.5% BBM medium in a glass aquarium (15×15×20 cm) for 18 days. At the 6th day of cultivation, they were treated with the addition of 100 ?M of Fe2+ and without the addition of Fe2+ (control). Each treatment was repeated 3 times. Growth measurement was carried out by weighing of fresh weight which was carried out every 3 days. On the 8th day of cultivation, 0.2 g of fresh weight of each sample was taken and then RNA isolation was performed using the TRIZOL method. The isolated RNA was then analyzed by Electrophoresis and NanoDrop Spectrophotometer methods, followed by cDNA synthesis that was carried out using GoTaq qPCR Master Mix. The cDNA was then amplified using the Real Time PCR method to test the gene expression of BKT (?-carotene ketolase) and CrtR-B (?-carotene hydroxylase). Culture of Spirogyra sp. reached the peak of growth at the 6th day of cultivation with an average fresh weight of 11.44g/L and gradually decreased in both control and treated cultures. The results of the fresh weight measurement in the control culture on the 9th day to the 18th day were 10,97 g/L; 6,05 g/L; 4,54 g/L and 2,32 g/L while in the Fe2+ stress treated-culture were 5,62 g/L; 2,85 g/L; 2,03 g/L and 0 g/L, respectively. This results indicated that the fresh weight of treated culture was lower compared to the control. The rate of decrease in the fresh weight of the treated culture on the day -9 to the day-18 were 48.78%, 52.89%, 55.15%, and 100% of the control culture, respectively. The results of the gene expression analysis for BKT and CHY/CrtR-B genes in the treated culture were 0.057 and 0.405 times the genes expression in the controled culture, respectively, which was smaller than the control culture. In conclusion, the addition of Fe2+ stress reduce the expression level of BKT (?-carotene ketolase) and CrtR-B (?-carotene hydroxylase) genes.