Preparation of a complementary DNA for leghaemoglobin and direct demonstration that leghaemoglobin is encoded by the soybean genome

Abstract

In soybean root nodulea, leghaemoglobin (Lb) accounts for 25–30% of the total soluble protein but is not detected in other tissues. In order to determine whether the Lb genes are plant or bacterial in origin a cDNA probe for Lb was prepared from 9S poly (A) containing mRNA of root nodules. Although this 9S mRNA directed synthesis of predominantly three forms of Lb in vitro the kinetics of hybridisation of cDNA and the 9S mRNA showed a transition at about 30% hybridisation which suggested that the 95-cDNA was not pure Lb-cDNA. The abundant, Lb-cDNA was prepared by two cycles of hybridising 9S mRNA and cDNA to a Rot of 3 × 10⁻³ and isolation of the hybridised cDNA on hydroxyapatite. The Lb-cDNA was homogeneous in hybridisation analysis with 9S mRNA and electrophoresis in 98% formamide gels. This cDNA hybridised with soybean DNA and not with Rhizobium DNA, thus directly demonstrating that Lb genes are of plant origin. Titration of Lb-cDNA with soybean DNA showed that Lb genes are reiterated about forty-fold per haploid genome.