Kloning Gen Proteinase Inhibitor dari Kulit Buah Kakao pada Vektor Ekspresi dengan Metode Gateway

Abstract

Cacao (Theobroma cacao L.) is one of a high economical value estate crops as an export commodity. Eighty percent of cacao production loss was caused by Cocoa Pod Borer (CPB), Conopomorpha cramerella Snellen. Thus, cacao seed were not soldable due to its low quality. Proteinase Inhibitor (PIN) is an antinutrition substances which able to inhibit proteolytic enzyme from insect gut. A 21 kDa of cacao seed PIN gene was known of having high homology to soybean trypsin inhibitor. This research was aimed to clone PIN gene on expression vector by Gateway method. Gateway method step include design Gateway primer, amplification PIN gene with Gateway primer, recombination PIN gene on Entry and Destination vector, transformation on Escherichia coli XL- 1 Blue, confirmation PIN gene on Entry and Destination vector, and transformation PIN gene on E.coli into Agrobacterium tumefaciens. Result of research indicate that PIN gene have a measurement of 850 bp succeed inserted into expression vector with Gateway method easy and quickly. The recombinant DNA was successfully transformed into Agrobacterium tumfaciens AGL-0 strain.