CONSTRUCT AND PRODUCTION GONAD-INHIBITING HORMONE dsRNA of BLACK TIGER PRAWN (Penaeus monodon) by in vitro and in vivo Technique in L4440 Expression Vector

• Main Author: Shestita Wulandari, Anggi
• Format: Theses
• Language: Indonesia
• Online Access: https://digilib.itb.ac.id/gdl/view/34869
• Institution: Institut Teknologi Bandung

Black tiger prawn (Penaeus monodon) is one of the valuable aquaculture comodities from Indonesia with great demand in the world that increases every year. Thereby increasing Penaeus monodon egg production in hatchery become more important nowadays. A conventional technique that had been used for years is eyestalk ablation by which gonad maturation process is stimulated. This technique, however, causes the broodstock can not be used for more than two cycles beside the decrease of egg quality. One approach to overcome this problem is RNA interference (RNAi), a technology that can inhibit Gonad Inhibiting Hormone (GIH) expression to stimulate gonad maturation. RNAi technology was done by producing GIH double-stranded RNA (dsRNA) by in vitro and in vivo techniques. Production of dsRNA started with total RNA isolation from eyestalk and reverse-transcribed by iScriptTM cDNA Synthesis Kit. GIH fragment was generated by designing 3 pairs of primer in small-interfering RNA (siRNA) potential region. Those three pairs of primer was used to amplify GIH fragment by touchdown PCR. Production of in vitro dsRNA was conducted by using MEGAscript® RNAi kit. A 271-bp GIH dsRNA was injected to two 12-14 months old female prawns. However, the injection did not accelerate spawning, probably related to the low dossage. Therefore, we need to produce large scale GIH dsRNA by in vivo method. GIH fragment that generated by touchdown PCR was ligated to pGEM® T-Easy cloning vector. The fragment was restristed with Not1 restriction enzyme and then ligated to L4440 expression vector. HT115 strain E.coli then transformed with the expression vector. Expression of GIH dsRNA in bacteria was induced by IPTG and then isolated by modified TRIzol method. A ±750-bp band proved that GIH dsRNA has been isolated from transfomant bacteria. Sequencing and reverse-transcription PCR also confirmed that GIH dsRNA has been succesfully constructed in L4440 expression vector.