TRANSIENT EXPRESSION ANALYSIS OF GUS REPORTER GENE IN TOMATO FRUITS DRIVEN BY Ma-ACS1 GENE PROMOTER FROM BANANA (Musa acuminata, AAA GROUP)

• Main Author: Meitha, Karlia
• Format: Theses
• Language: Indonesia
• Online Access: https://digilib.itb.ac.id/gdl/view/34919
• Institution: Institut Teknologi Bandung

Producing recombinant protein in plants (such as edible vaccine) will allow crop plants to deliver important protein for the consumer and the price is estimated cheaper since no industrial unit is needed in the proteins production. Fruit is a perfect organ to facilitate recombinant proteins delivery to human, because this is the most consumed plant organ. In achieving this aim, the availability of a specific gene promoter for regulating gene expression in fruit is highly required. Ma- ACS1 (ACC synthase1) gene promoter from banana is a promising candidate to fulfill the need. Since the gene is specifically expressed in fruit during ripening phase. The ACS gene is responsible in synthesizing ACC (1-aminocyclopropane- 1-carbocylic acid), which further will be oxidized by ACC oxidase and result in ethylene. This research was aimed to isolate Ma-ACS1 gene promoter from banana (Musa acuminata AAA Group) cultivar Pisang Ambon Lumut and analyze its ability in transiently driving GUS gene expression in tomato fruits. Fragment isolation was conducted by PCR and confirmed by sequencing analysis. The isolated Ma-ACS1 gene promoter is 1557 bp long and characterization result showed that the promoter contains Elicitor Responsive Element (-1240 to -1235), Ethylene Responsive Element (-211 to -206), CAAT box (-150 to -147), TATA box (-33 to -27), and 5`Untranslated Region (1 to 73). The existence of elicitor responsive element (EIRE) and ethylene responsive element (ERE) has made Ma- ACS1 gene promoter as an inducible one. Because EIRE is inducible by the presence of fungal and yeast elicitor, while ERE is inducible by ethylene. Ma- ACS1 gene promoter was then constructed in binary vector pCambia 1304 to regulate reporter gene (GUS) expression. Construction was performed by restriction-ligation method and plant transformation by agroinjection method to ripe and unripe tomato fruits. GUS histochemical assay result showed that Ma- ACS1 gene is able to drive GUS gene expression transiently in ripe and unripe tomato fruits. However, the expression level was higher in unripe fruit due to more plant cells were transformed rather than in ripe one. This was caused by tissue softening in ripe fruit has lead to reduction of vascular bundles ability in transporting A. tumefaciens cells to spread thoroughly inside the fruit.