Cloning of ndhK from soybean chloroplasts using antibodies raised to mitochondrial complex I

Abstract

A soybean shoot cDNA expression library was screened with polyclonal antibodies raised against red beet complex I and several clones were identified. One clone, consisting of a 1 kb insert, was fully sequenced. The sequence of 1025 bp was found to contain two extended open reading frames and the proteins encoded were identified as the ndhK and ndhJ products of the chloroplast genome. Nuclear, mitochondrial and chloroplast DNA was isolated and probed with a ndhK-specific probe. The chloroplast DNA contained a single copy of the cloned insert. With nuclear DNA, positively hybridising bands of 1.2, 2.7 and 3.2 kb were observed indicating that at least one gene homologous to ndhK of the chloroplast genome, is also present in the nucleus. The mitochondrial DNA did not hybridise with the ndhK probe. Western analysis of thylakoid proteins with the mitochondrial complex I antibodies revealed several bands. It is suggested that soybean contains two copies of the ndhK gene, one, on the plastid genome, coding for a subunit of a chloroplast NAD(P)H dehydrogenase, and the other, in the nucleus, coding for a subunit of mitochondrial complex I.