Molecular cloning, expression profiling, and yeast complementation of 19 beta-tubulin cDNAs from developing cotton ovules

Abstract

Microtubules are a major structural component of the cytoskeleton and participate in cell division, intracellular transport, and cell morphogenesis. In the present study, 795 cotton tubulin expressed sequence tags were analysed and 19 beta-tubulin genes (TUB) cloned from a cotton cDNA library. Among the group, 12 cotton TUBs (GhTUBs) are reported for the first time here. Transcription profiling revealed that nine GhTUBs were highly expressed in elongating fibre cells as compared with fuzzless-lintless mutant ovules. Treating cultured wild-type cotton ovules with exogenous phytohormones showed that individual genes can be induced by different agents. Gibberellin induced expression of GhTUB1 and GhTUB3, ethylene induced expression of GhTUB5, GhTUB9, and GhTUB12, brassinosteroids induced expression of GhTUB1, GhTUB3, GhTUB9, and GhTUB12, and lignoceric acid induced expression of GhTUB1, GhTUB3, and GhTUB12. When GhTUBs were transformed into the Saccharomyces cerevisiae inviable mutant, tub2, which is deficient in beta-tubulin, one ovule-specific and eight of nine fibre-preferential GhTUBs rescued this lethality. This study suggests that the proteins encoded by cotton GhTUBs are involved during cotton fibre development.

Keywords: Gossypium hirsutum (cotton) fibre; fuzzless-lintless mutant; phytohormone; β-tubulin.

Fig. 1.

Alignments and neighbor-joining tree of the predicted amino acid sequences encoded by cotton GhTUB genes. (A) Amino acid sequences at the N termini of 19 cotton TUBs are aligned. The conserved residues are shaded in black. (B) A neighbor-joining tree was constructed in MEGA3.1 from 1000 bootstrap replicates. The scale bar corresponds to 0.1 estimated amino acid substitutions per site. GhTUB1, GhTUB3, GhTUB5, GhTUB6, GhTUB7, GhTUB9, and GhTUB12 were from GenBank (respective accession numbers: AY345610, AY345606, AY345607, AY345608, AY345609, AF521240, DQ023526). The other GhTUBs were from this study.

Fig. 2.

RT-PCR analysis of 19 GhTUB transcripts in wild-type and fl mutants. (A) RT-PCR analysis of nine GhTUB transcripts expressed preferentially or differentially in fibres. (B) RT-PCR analysis of nine GhTUB transcripts that were not preferentially expressed in fibres. Dpa, Days post-anthesis; WT-0, 0 dpa wild-type ovules; WT(F)-10, 10 dpa wild-type fibres stripped from ovules; fl-0, 0 dpa fl mutant ovules; fl-10, 10 dpa fl mutant ovules; R, roots; S, stems; L, leaves. UBQ7 (GenBank accession no. AY189972) was included as a control.

Fig. 3.

Exogenous phytohormones and lignoceric acid promote the accumulation of GhTUB transcripts. RNA samples were prepared from three independent ovule cultures in the presence or absence of 5 μM brassinosteroid (BR), 0.1 μM ethylene (ETH), 5 μM gibberellin (GA), and 5 μM lignoceric acid (C24:0) for the indicated times. QRT-PCR experiments used the gene-specific primers reported in Table 1. The relative expression level of each GhTUB transcript is expressed relative to the level of the same transcript in the medium with or without ∼0.8 μM methyl tert-butyl ether for lignoceric acid supplementation at each indicated time. Values <0.1 are not shown. Statistically significant differences were determined using one-way ANOVA combined with Tukey's test. ***, P <0.001; **, P <0.01; *, P <0.05.

Fig. 4.

Certain GhTUBs restore the viability of S. cerevisiae tub2Δ haploid cells. (A) Tetrad from diploid cells W1536 TUB2/tub2Δ transformed with pYADE4-ScTUB2. The ascospores grown on the YPD plate were replicated to G418 and FAA plates. (B) Complementation of tub2Δ mutant cells by individual GhTUBs. 12 h, RT-PCR analysis of yeast mutant cells expressing different GhTUBs harvested at 12 h from Sc-Trp culture medium. One S. cerevisiae actin gene (Act1) was used as the template control of RT-PCR analysis.