CONFIRMATION OF HEAT STRESS ELEMENT (HSE) SEQUENCES IN METUF C PROMOTER OF CASSAV (MANIHOT ESCULENTA CRANTZ.) VAR. ADIRA

• Main Author: Yusva, Humaira
• Format: Final Project
• Language: Indonesia
• Online Access: https://digilib.itb.ac.id/gdl/view/37119
• Institution: Institut Teknologi Bandung

Metuf C promoter was isolated from Metuf gene family of cassava (Manihot esculenta Crantz.) plant cultivar Adira. Basically, Metuf C express EF-Tu protein (Elongation Factor-Thermo unstable) that finds to function in elongation factor and as molecular chaperon. One component that plays an important role in this promoter is HSE (Heat Stress Element) that is responsible as the binding site of HSF (Heat Stress Transcription Factor). In the previous study, Metuf C promoter was cloned and then constructed on pBI121. The construct was analyzed by transient transformation in tobacco (Nicotiana tabacum L.). However, as a result, Metuf C gene that codes ?-glukuronidase enzyme (GUS) was not expressed in tobacco. Hypothetically, this is due to one-base difference in alignment result of HSE from M. esculenta var. Adira and M. esculenta var AM560-2 that was developed by International Center for Tropical Agriculture (CIAT), USA and used as the basic in database construction. Therefore, this study aims to conduct a confirmation of HSE in M. esculenta var Adira. The research was initiated by examination of transient transformation of Metuf C in tobacco using Agrobacterium tumefaciens AGL1 that was inserted with binary pBI121-Metuf C, pBI121-CaMV35S (positive control), and Agrobacterium tumefaciens without binary vector (negative control). Histochemically, only CaMV35S that regulates the expression of GUS gene. Based on that result, confirmation was conducted from isolated genome DNA of M. esculenta by electrophoresis and PCR. Metuf C was isolated using specific primer to produce amplicon and then, the amplicon was purified and ligated to cloning vector pJET1.2/blunt. The result of ligation was transformed to E.coli DH5? and followed by isolation and confirmation using PCR and also comparing it with sequencing result of Mitryadinillah (2016). Metuf C promoter was successfully cloned in pJET1.2/blunt that was shown by size of amplicon (±1600 pb). The identity percentage of sequence alignment was 99.9% using Metuf C Mitryadinillah (2016) promoter. This result proved there is no difference between HSE sequences but the differences were only detected in the gap and one base in four bases after HSE sequence.