Differential regulation of six novel MYB-domain genes defines two distinctexpression patterns in allotetraploid cotton (Gossypium hirsutum L.)

A PCR-based strategy was employed to identify myb-related genes potentially involved in the differentiation and development of cotton seed trichomes. cDNA clones representing six newly identified cotton myb-domain genes (GhMY B) of the R2R3-MYB family were characterized in the allotetraploid species Gossypium hirsutum L. (2n = 4x = 52; AADD). Several interesting motifs and domains in the transregulatory region (TRR) were identified as potential ca ndidates for modulating GhMYB activity. One such structural feature is a ba sic 40-amino acid stretch (TRR1) located immediately downstream of the DNA- binding domain (DBD) in five of the GhMYBs. Furthermore, the conserved moti f GIDxxH identified in a subset of plant MYBs is also present in the same p osition in the TRR1 domains of GhMYB1 and GhMYB6, exactly 12 amino acid res idues downstream of the last tryptophan in the R3 repeat of the DBD. At lea st two of the GhMYBs (GhMYB4 and GhMYB5) contain unidentified ORFS in the 5 ' leader sequence (5'-uORFs) that may serve to regulate the synthesis of th ese particular GhMYB proteins at the translational level. Multiple alignmen t of DBD sequences indicated that GhMYBs show structural similarity to plan t R2R3-MYB factors implicated in phenylpropanoid biosynthesis. GhMYB5 is th e most distantly related cotton R2R3-MYB and is found in an isolated cluste r that includes the drought-inducible AtMYB2. Sequence comparisons of DBD a nd TRR domains from GhMYBs, MIXTA (AmMYBMx) and G11 (AtMYBG11) did not reve al any striking similarity beyond conserved motifs. However based on earlie r phylogenetic analysis, GhMYB2, GhMYB3. and CHMYB4 are members of a cluste r that contains GLABROUS1, while GhMYB1 and GhMYB6 belong to a closely rela ted cluster. Semi-quantitative RT-PCR analysis revealed two discrete patter ns of GhMYB gene expression. Type I cotton MYB (GhMYB-1, -2, and -3) transc ripts were found in all tissue-types examined and were relatively more abun dant than these derived from type II GhMYB genes (GhMYB-4, -5, and -6), whi ch showed distinct, tissue-specific expression patterns. The developmental regulation of GhMYBs is consistent with a role for these DNA-binding factor s in the differentiation and expansion of cotton seed trichomes.