Cloning, characterization and expression analysis of <em>APETALA2</em> genes of <em>Brassica juncea</em> (L.) Czern.

Abstract

The APETALA2/Ethylene-Responsive Factor (AP2/ERF) is one of the largest gene families encoding several plant specific transcription factors. It plays significant roles in growth and development process, biotic and abiotic stresses, and responses to hormones. <em>AP2</em> is a homeotic gene governing floral meristem specification, floral organ determination and floral homeotic gene expression in <em>Arabidopsis.</em> The basic structure of <em>AP2</em> gene was unchanged during evolution in diploid species. The present study was undertaken to find whether <em>AP2</em> has undergone any change in structure or expression pattern during evolution of allopolyploid <em>Brassica juncea</em>. We cloned <em>AP2</em> orthologs and c-DNAs from <em>B. juncea</em> and <em> B. nigra</em>. <em>B. juncea</em> was found to carry three <em>AtAP2</em> orthologs. Comparison of <em>BjAP2</em> genes with <em>AP2</em> orthologs from progenitor species, <em>B. rapa</em> and <em> B. nigra</em> showed that two of the <em>BjAP2</em> genes were derived from <em>B. rapa</em> and one from <em>B. nigra</em>. <em>BjAP2</em> genes have retained its characteristic AP2 domain and <em>miR172</em> complementary sequences. mRNAs originated from three <em>AP2</em> orthologs were detected in all the tissues examined, namely, leaf, flower buds and seedling, indicating absence of sub-functionalization of <em>AP2</em> during polyploid evolution. However, one of the <em>B. rapa</em> copies gave alternatively spliced <em>AP2</em> transcript which lacked the second exon. Consequently, the splice variant could not be translated into functional AP2 protein. Considering that <em>miR172</em> suppresses translation of <em>AP2</em> transcripts, the alternatively spliced transcript could still play important regulatory role by limiting the availability of <em>miR172</em> molecules to bind to functional <em>AP2</em> transcripts. qRT-PCR analysis of <em>BjAP2</em> expression in different accessions of <em>B. juncea</em> with contrasting seed size indicated that <em>BjAP2</em> is not a major determinant of seed size in mustard.