Reverse transcription and kinetic complexity analysis of mRNA from lupin root nodules

Abstract

Complementary DNA (cDNA) synthesis was performed using mRNA from lupin root nodules and from lupin uninfected roots as templates. Optimal conditions for the synthesis of 700 nucleotides long cDNA were established. Hybridization kinetics of mRNA-cDNA were performed in a homologous system from root nodules as well as in a heterologous system. Hybridization analysis revealed the presence of four frequency abundance classes within the root nodule mRNA population. In order to enrich the cDNA population in leghaemoglobin sequences, two synthetic pentadecanucleotide fragments complementary to the putative 3' ends of leghaemoglobin mRNAs were used as primers for the reverse transcription of nodule poly(A)-RNA. The resultant cDNA was enriched in rapidly hybridizing components which may contain leghaemoglobin sequences.