A translational genomics approach to pod specific promoter isolation in Theobroma cacao

Cocoa pod borer (CPB) or Conopomorpha cramerella (Snellen) is the most damaging CPB pest of cocoa in Malaysia. It can cause complete crop loss unless measures are taken to control it. CPB is considered the major hindrance to the development of the cocoa planting industry (Lim, 1992). The production of cocoa dry beans in Malaysia in 2005 is 27,964 tonnes (Malaysian Cocoa Statistics, 2006). Average bean losses in Malaysia due to CPB have been estimated to be at least 15 percent of annual potential yields. This amounted to approximately RM20,973,OOO in losses at the current market price. Currently, the most common control measure is spraying with insecticides coupled with cultural practices. Pesticides are expensive, often misused and ineffective against larvae within pods, lead to resistant insects, kill beneficial insects and leave residues in beans and negative effects on the environment. Planting resistant trees would be the best and cheapest approach to prevent damage by CPB. Thus far, no CPB-resistant cocoa trees have been identified. An alternative would be to genetically engineered cocoa to produce proteins that are toxic to CPB larvae and not to humans and other animals. Cocoa pod is the most economic part of the plant. In order express the insecticidal protein in the pod shell instead of the seeds, it is crucial to target the expression of these proteins in the pod. Therefore a gene promoter that specifically drives the expression of the insecticidal gene(s) in the pod tissue is necessary in the development of transgenic cocoa plant resistant to CPB. The study here uses a translational genomics approach to isolate pod specific promoter. Arabidopsis microarray data mining was carried out to identify genes that specifically expressed in the fruit tissues of the plant. These gene sequences are collected and subsequently blasted against cacao expressed sequence tags (ESTs) database. Those matching sequences ar e identified and primers are designed. Probes are amplify by polymerase chain reaction (PCR) using these primers. Total RNA are isolated from various tissues of the cocoa plant. The probes generated are then used for Northern analysis with these RNA isolated. Two putative cocoa pod-specific genes have been discovered; pectin methylesterase and GDSL-motif. Cocoa bacteria artificial chromosome (BAC) library is screened with these probes. Seven BAC clones were identified. These clones will be isolated and sequencing upstream will be carried out to clone the promoter region. The gene promoter will be fused with a reporter gene and transformed into Arabidopsis and T. cacao. Finally, functional analysis of the plant will be carried out to determine the exact tissues of the expression.