Cloning and activity analysis ofin vitro expression of plant NAD-IDH genes

Abstract

The plant NAD-dependent isocitrate dehydro-genase (NAD-IDH) is an important multifunctional enzyme. The cDNAs encoding three different subunits of the NAD-IDH were cloned successfully from leaves of Arabidop-sis thaliana ecotype Columbia gl1 and three lines (Shaan 2A, Shaan 2B and Ken C1) of Brassica napus by RT-PCR method. By searching the sequences in GenBank Database, it is shown that these sequences from B. napus were novel. Their encoding regions of a functional protein were then inserted into the multiple cloning sites of pMID1 vector for the expression of recombinant protein. All the recombinants were successfully expressed in the in vitro expression system. When the transcripts of subunits 0, 1 and 2 were added to-gether to the in vitro system with 36 靏/mL of protein disul-fide isomerase, the expressed products had NAD-IDH enzy-matic activity. Comparison of different kinds of gene and molecular ratio of the transcripts showed that the NAD-IDH enzyme is composed of three subunits designated subunit 0, subunit 1 and subunit 2. All the three subunits are essential to catalytic activity. Missing subunit 0 could abolish activity. Missing either subunit 1 or subunit 2 would cause severe impact on activity. Deletions, which would cause frameshift mutation or nonsense mutation, were also found in some transcripts of subunit 1 gene from Shaan 2A and Shaan 2B of B. napus. The mutated subunit 1 lost its proper function. This may explain why there is difference of the NAD-IDH activity among three lines of B. napus.