Cloning, Characterization, And Functional Analysis Of MYB Transcription Factors Gene From Cotton

Cotton (Gossypium hirsutum L.) is an important economic crop that is extensively usedin the textile industry and as. Cotton fibers are derived from single-celled, epidermaltrichomes that develop from the protodermal layer of maturing seed. A large number ofstudies showed that the cotton fiber and Arabidopsis thaliana tricome is originated in theepidermal cells of single cell, and they are likely to the similar development mechanism.A lot of genes are required for fiber differentiation and development, but so far, little isknown about how these genes control and regulate the process of fiber development. MYBtranscription factor, one of the most important protein families in plants, is involved in theregulation of secondary metabolism, morphogenesis of plant, responding to environmentstress and plant hormone.GL1as a R2R3-MYB transcription factor play a impotant role inArabidopsis thaliana tricome development, so we used this sequence as reference to get twocotton sequences by tblastn program from a D5chromosome library of G. raimondii. In orderto explore their expression patterns in upland cotton, they were cloned from Xuzhou142byRT-PCR. One named GhMYB0and the other named GhMYB2-like as a new isoform ofGhMYB2in wild type Xuzhou142. The experimental results were showed as follows:1. By using the protein sequence of GL1as reference, through the electronic cloning andRT-PCR, we successfully cloned two MYB family genes with the whole length of the genecDNA from upland cotton, named GhMYB0and GhMYB2-like. The open reading frame ofGhMYB0is843bp in length, which encodes280aminoacid residues. GhMYB2-like cDNAcovered381bp, and encoded a polypeptide of126amino acids. Compared with the GhMYB2this sequence is deprived215bp of the sequence from254to469.2. Motif search results indicated that GhMYB0is belonging to the R2R3-MYB family.Compared with GhMYB2, the deprivation region of GhMYB2-like was R3domain andGhMYB2-like protein contained typical Rl/2subfamily motif MYB family. Homology blastand phylogenetic tree analyses showed that GhMYB2-like has51%similarity with AtMYB0.3. The expression pattern analysis of different stages of fiber development and organsrevealed that GhMYB0was highly expressed in flowers, then ovules, as well as highlyexpressed from0dpa, and reached at the highest level on20dpa (days post anthesis). GhMYB2-like showed the highest expression level in ovule at0dpa. Further, the expressionlevel in Xuzhou142wild type was higher than mutant of Xuzhou142.4. GhMYB0over-expressed lines of Arabidopsis exhibited fewer trichomes than the wildtype, and this gene can restore the notrichome phenotype of gl-1mutant of Arabidopsis. Inaddition, the transgenic lines were shorter plant heights, longer vegetative growth time, andpollen abortion.Overexpressing GhMYB2-like in Arabidopsis thaliana Columbia-0(Col-0)activated fiber-like hair production in5.98%of seeds and had no obvious effects on trichomedevelopment in leaves or siliques.3transgenic plant lines from T2generation were randomlyselected, and qRT-PCR analyses showed that the endogenous gene AtMYB0of Arabidopsisthaliana expression quantity was increased result from the cotton GhMYB2-like gene.Our results showed that GhMYB0and GhMYB2-like are required for cotton fiberinitiation and development, as well as reveal a largely conserved mechanism of theR2R3-MYB transcription factor in cell fate determination in plants.