Molecular characterization of the cotton GhTUB1 gene that is preferentially expressed in fiber

Abstract

Each fiber of cotton (Gossypium hirsutum) is a single epidermal cell that rapidly elongates to 2.5 to 3.0 cm from the ovule surface within about 16 d after anthesis. A large number of genes are required for fiber differentiation and development, but so far, little is known about how these genes control and regulate the process of fiber development. To investigate gene expression patterns in fiber, a cDNA, GhTUB1, encoding beta-tubulin was isolated from a cotton fiber cDNA library. The analyses of RNA northern-blot hybridization and reverse transcriptase-polymerase chain reaction demonstrated that GhTUB1 transcripts preferentially accumulated at high levels in fiber, at low levels in ovules at the early stage of cotton boll development, and at very low levels in other tissues of cotton. The corresponding GhTUB1 gene including the promoter region was isolated by screening a cotton genomic DNA library. To demonstrate the specificity of the GhTUB1 promoter, the 5'-flanking region including the promoter and 5'-untranslated region was fused with the beta-glucuronidase reporter gene. The expression of the reporter chimera was examined in a large number of transgenic cotton plants. Histochemical assays demonstrated that GhTUB1::beta-glucuronidase fusion genes were expressed preferentially at high levels in fiber and primary root tip of 1- to 3-d-old seedlings and at low levels in other tissues such as ovule, pollen, seedling cotyledon, and root basal portion. The results suggested that the GhTUB1 gene may play a distinct and required role in fiber development. In addition, the GhTUB1 promoter may have great potential for cotton improvement by genetic engineering.

Figure 1

Northern analysis of GhTUB1 transcripts in cotton fiber, ovule (seed), petal, anther, leaf, stem, cotyledon, and root. Total RNA (20 μg lane⁻¹) from leaf (1), stem (2), root (3), cotyledon (4), petal (5), anther (6), 4, 8, 14, 21, and 28 DPA ovule (7–11; young seed), and 8 and 14 DPA fiber (12 and 13) was fractionated on a denaturing 1.2% (w/v) agarose gel and transferred to nylon membrane (see “Materials and Methods”). A, Autoradiograph of RNA hybridization; B, loading of total RNA (20 μg lane⁻¹) fractionated on a denaturing 1.2% (w/v) agarose gel.

Figure 2

GhTUB1 gene structure and construction of the chimeric genes between the GhTUB1 5′-flanking region and the GUS gene. The physical structure of the GhTUB1 gene was characterized in detail. GhTUB1 encodes a 445-amino acid β-tubulin polypeptide. The translated portions of exons are denoted by black boxes; introns, the 5′-flanking region (including putative promoter and 5′-UTR), and the 3′ terminus are denoted by lines. The lengths of the introns in base pairs are indicated. The number at the boundaries of each exon indicates the codon at which the intron is located. Intron 1 splits codon 132, and intron 2 splits codon 222. The translation initiation and translation termination codons are shown. The length of the 5′-flanking region and cloning sites used for fusion constructs are shown. A, GhTUB1 gene structure; B, GhTUB1::GUS fusion; aa, amino acids.

Figure 3

Genomic DNA Southern-blot analysis. Total genomic DNA (30 μg lane⁻¹) digested with restriction enzymes BamHI (B), EcoRI (E), EcoRV (EV), and SacI (S) and fractionated on a 0.8% (w/v) agarose gel was blotted to nylon membranes and hybridized with ³²P-labeled GhTUB1 5′-region gene-specific probe (A) and ³²P-labeled GhTUB1 ORF (third exon) probe (B).

Figure 4

Histochemical localization of GUS gene expression at the early stage of the fiber development in transgenic plants containing the GhTUB1/GUS fusion gene. A through D, Micrographs of 5-μm-thick cross sections of 1 to 2 DPA ovules. A and B, Early and late of 2-DPA ovules (250× and 700×, respectively) of transgenic cotton with 1.4-kb GhTUB1::GUS fusion. High level of GUS activity was only found in the fiber, and very weak GUS staining was seen in the inner cell layers of seed coat in some transgenic lines, but no GUS expression was detected in the outermost cell layer except fiber cells. C and D, One- and 2-DPA ovules (500× and 350×, respectively) of transgenic cotton with 0.9-kb GhTUB1::GUS fusion. Strong GUS activity was observed in the fiber and in the inner cell layers of the seed coat, but no GUS staining was detected in the outermost cell layer (the epidermis) except fiber cells. In all of the transgenic ovules, GUS activity was at an undetectable level in the embryo.

Figure 5

Histochemical localization of GUS gene expression in transgenic cotton plants containing the GhTUB1/GUS fusion gene. A through C, One-, 2-, and 3-DPA ovules (50×). Strong GUS activity was observed in the fiber. D, Fourteen-DPA boll (cross section) of transgenic cotton with 1.4-kb GhTUB1::GUS fusion. Strong GUS activity was seen in the developing fiber. E, Fourteen-DPA boll (cross section) of transgenic cotton with 0.9-kb GhTUB1::GUS fusion. Strong GUS activity was detected in the developing fiber, and weak GUS staining was also visible in the seed coat and the embryo. F, Flower bud (longitudinal section, 5×) of transgenic cotton with 1.4-kb GhTUB1::GUS fusion. GUS activity was undetectable in the tissues of the bud. G, Flower bud (longitudinal section, 5×) of transgenic cotton with 0.9-kb GhTUB1::GUS fusion. Moderate or strong GUS activity was observed in the pollen, and weak or moderate GUS staining was found in the ovary and style. H through J, Three-, 7-, and 10-d-old seedlings of transgenic cotton with 1.4-kb GhTUB1::GUS fusion. H, Three-day-old cotton seeding. GUS gene was expressed moderately or weakly in the cotyledon and root basal portion, and high level of GUS expression was located in the primary root tip. I, Seven-day-old cotton seedling. Weak GUS activity was observed only in the cotyledon. J, Ten-day-old cotton seedling. GUS activity was decreased to a very low level in the cotyledon, and no GUS expression was detected in the leaf and shoot apex. K, From left to right: leaf, stem, root, sepal, and petal of transgenic cotton plant. No GUS activity was found in all of the tissues.