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 [9]. 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 [10] in DNAStar software (DNAStar, WI, USA), and the phylogenetic trees were viewed by TREEVIEW [11] programs.
