The Function Of GhTUB7 In Cotton Fiber Development

Cotton is the most important fiber crop in the world. Cotton fibers, the main products of cotton, are highly elongated single cells from epidermal cells of cotton ovules. The development of cotton fiber includes four well-defined, yet overlapping stages: initiation, elongation, secondary cell wall accumulation and maturation. Research on fiber cell development has significance and practical value to cultivate high yield and quality cotton. Since fiber cell has several defined developmental stages and two of them last for a long time, cotton fiber regards as an ideal model for studies of plant cell elongation and cell wall biogenesis.Microtubules are important parts of the cytoskeleton, play key roles in plant cell development. To adapt to the complicated developmental changes, cotton fiber cell needs to apply special approaches to coordinate the arrangement of the microtubule pattern. But the mechanisms of MT arrangement and regulation in fiber cell development are largely unknown. Microtubules are formed by the self-assembly of α-β tubulin heterodimers. Many α- and β-tubulin genes, whose sequence were highly conserved, were found in plant genome. Some genes show tissue-specific/preferential and developmental stage expression, suggesting that these genes play diverse roles in the plant cell growth and development. Some tubulin genes were preferential expressed in fiber cell, intimating they were involved in fiber cell development, though the detail functions were not clear. Functional characterization of these genes were the basic works to illuminate the regulatory mechanism of microtubule in fiber development.To date, 38 β-tubulins were found in upland cotton genome(Gossypium hursutum L.). A beta-tubulin gene named Gh TUB7, was preferential expressed in fiber cell. High expression level of Gh TUB7 occurred at the secondary cell wall accumulation stage suggesting that Gh TUB7 was related to the quality of fiber. For better understanding the function of Gh TUB7, this gene and its promoter were cloned and characterized. In this study, the approaches of up- and down-regulating the expression level of Gh TUB7 were applied to investigate the role of Gh TUB7 in the growth and development of cotton fiber. The effects of Gh TUB7 on regulating fiber growth and development were investigated by phenotype analysis of Gh TUB7 transgenic cotton fibers. The function of promoter was characterized to better understand the regulatory mechanism. This study will provide some data for the mechanism of fiber cell growth. The following results are obtained.1. Fiber-specific gene Gh TUB7 was cloned from cotton The length of cloned c DNA fragment was 1471 bp, containing a 1335 bp ORF encoding 444 amino acid residues. The weight of the protein was 50 k D, and its isoelectric point was 4.766. Protein sequence analysis showed this protein containing tubulin domain. High homology was found with other β-tubulins from Arabidopsis thaliana, Oryza sativa, Theobroma cacao and Glycine max. Phylogenetic relationship analysis showed that Gh TUB7 had a higher similarity with Theobroma cacao.2. Expression Patterns of Gh TUB7 Digital expression profile and RT-PCR results show that Gh TUB7 is a preferential expressed gene in fiber. The transcript of Gh TUB7 was detected only in fiber cell while hardly detected in the sample of root, stem, leaf and ovule. The transcript reached high level at 16 DPA and gradually increased following fiber development. The highest expression level displayed on 20 DPA and then declined slightly on 22 DPA. High expression of Gh TUB7 occurred at secondary cell wall stage suggesting that Gh TUB7 play important roles in secondary cell wall accumulation.3. Cotton fiber-specific promoter Gh TUB7 P Gh TUB7 is a fiber-specific gene and high expression level was detected at late developmental stage of fiber cell, suggesting that the research on the promoter Gh TUB7 P may have great value. To get the core sequence of promoter Gh TUB7 P, 5’-deletion experiments of promoter were performed when the upstream regulatory sequence of 2010 bp of the gene was cloned. For the visualization strategy, promoter sequences were fused with GUS report gene. GUS staining results showed that in Gh TUB7P::GUS cotton, the GUS activity was detected only in fiber cell while hardly detected in the samples of root, stem and leaf. We also checked the GUS activity at various developmental stages of fiber. GUS signal was hardly detected in the 10 DPA fiber and gradually increased following fiber development. Strong GUS activity was observed in the 20 DPA and 30 DPA fiber cells, which were consist with the expression patterns of Gh TUB7 displayed by RT-PCR. We can conclude that the Gh TUB7 P is a strong fiber-specific promoter.4. Gh TUB7 affects the development of cotton fiber Fiber cell initiation and elongation were inhibited in overexpressing Gh TUB7 transgenic cotton. Mature fiber length of Gh TUB7 over-expressing transgenic cotton was 12.2% shorter, compared with wild type. Up-regulating the expression level of Gh TUB7 promoted the Ces As(Cellulose Synthase) expression level and added the thickness of cell wall. The treatment of tubulin inhibitor Colchicine suppressed the fiber cell elongation and this can be rescued by up-regulating the Gh TUB7 expression level. Contrarily, down-regulated the expression level of Gh TUB7 had no effect on fiber cell initiation, but it promoted fiber elongation. The transcript level of Ces A genes were lower when we down-regulated Gh TUB7 expression level, which might suppressed the secondary cell wall accumulation. We can conclude that Gh TUB7 was a key gene in fiber development and played a role in regulating the secondary cell wall accumulation.