Cloning strategies for the isolation of an a-amylase inhibitor/endochitinase gene from the seeds of Coix lachryma-jobi

Abstract

The aim of this work was to isolate the gene encoding a bifunctional a-amylase inhibitor/endochitinase protein from the seeds of Coix lachryma-jobi, a tropical cereal. Prior to this study, it had been demonstrated that this bifunctional protein had anti-insect and possibly anti-fungal properties. Consequently the gene could potentially be used to confer insect and fungal resistance in transgenic plants. A multifunctional approach was undertaken to isolate the a-amylase inhibitor/endochitinase cDNA and genomic sequences, involving three main strategies. Immunoscreening a Coix cDNA expression library with antibodies raised against a wheat germ endochitinase protein resulted in the isolation of three immunopositive clones. These cDNA's, were sequenced and one characterised as a seed storage protein, named a-coixin. Despite extensive searches of the appropriate databases, the function of the other two are as yet unknown. Another strategy was the production of polyclonal antibodies, raised against a glutathione S- transferase-a-amylase inhibitor fusion peptide. It was envisaged that these antibodies could be used to isolate the gene of interest following immunoscreening of the Coix cDNA expression library. Polyclonal antibodies were successfully elicited against the glutathione S- transferase moiety, but could not detect the a-amylase inhibitor protein when assayed. Using the polymerase chain reaction, amplification of the a-amylase inhibitor coding sequence was attempted from Coix genomic DNA, cDNA and a Coix seed cDNA library. PGR product were successfully amplified from genomic DNA and the cDNA library. Further characterisation of these product revealed that they were a result of non specific amplifications. Further work required to isolate the a-amylase inhibitor gene is discussed.