Cloning of flavonoid structural genes. To Palosuran
corresponding genes from cotton fiber, an Arabidopsis chalcone isomerase (CHI), a Malus flavanone 3-hydroxylase (F3H), a Daucus carota anthocyanidin synthase (ANS) and a cDNA-AFLP differential fragment ( Table 1) were used as probe sequences to query cotton ESTs in GenBank with the tBLASTn program (http://www.ncbi.nlm.nih.gov/blast). The homologous ESTs were assembled into contigs using SeqMan program of DNAStar software (DNAStar, WI, USA), and the contigs were subjected to BLASTX analysis (http://www.ncbi.nlm.nih.gov/blast) to find potential full-length ORFs. Subsequently, primers encompassing the putative ORFs were synthesized to amplify the cotton flavonoid structural genes using the cDNA derived from T586 fibers of 8–10 DPA as template ( Table 1). To clone
the anthocyanidin reductase (ANR) gene from cotton, the primers were designed according to the Gossypium arboretum BAN gene . The amplified cDNAs were cloned and sequenced. The predicted proteins were further used to perform homology search in GenBank using BLASTP program (http://www.ncbi.nlm.nih.gov/blast). To further characterize these cotton flavonoid structural genes, we performed multiple alignment and phylogenetic tree analyses using the predicted proteins and their homologs. The alignments were generated using CLUSTALW  in DNAStar software (DNAStar, WI, USA), and the phylogenetic trees were viewed by TREEVIEW  programs.