Cloning And Characterization Of Vacuolar H~+-ATPase Subunit C Gene From Gossypium Hirsutum

Cotton is the world's leading fiber crop. Cotton fiber is an elongated single cell of the ovule epidermis that undergoes rapid and synchronous elongation. Cotton fiber development consists of four discrete yet overlapping stages: initiation, elongation, secondary cell wall accumulation and maturation. During the rapid polar-expansion phase of development, fiber cell may elongate at peak rates in excess of 2.0mm per day. Therefore, fiber cell elongation is one of the important physiological processes directly determined final fiber length, which is an important trait in agronomy. However, the molecular base of fiber cell elongation largely unknown.Plant hormones are any of various hormones produced by plants that control or regulate germination, growth, metabolism, or other physiological activities. Brassionsteroids (BRs) is nominated as the "The Sixth Plant Hormone" of plant. Previous studies indicated that applied into the treatment of plant was able to improve the growth and productivity. Although overexpressing the gene involved BRs synthesis improve the quantity and quality of cotton fiber, less studies of the gene involved in BRs signal transduction through transgenic strategies have been reported up to now.Vacuolar H~+-ATPase (V-ATPase) was regarded as a key enzyme promoting the fiber cell elongation through regulating turgor-driven pressure involved in polar-expansion of single cell fiber. The DET3, encoding V-ATPase subunit C, played an important role in assembling the subunits of V-ATPase complex and regulating the complex activity, and involved in Brassinosteroids induced cell elongation. In order to understand the effects of cotton V-ATPase subunit C gene on fiber growth and development and the molecular base of BR action in fiber cell growth, we cloned the V-ATPase subunit C gene. Gene sequence analysis and expression pattern were taken, and then cotton and tobacco, as plant models, were transformed for genetic analysis.1. Identification and Characterization of GhDET3AtDET3 was utilized as electronic probe to screen the candidate sequences in NCBI database, and 3'-RACE technique was applied to clone the 3'-downstream sequence for the full length of GhDET3 cDNA. The full length clones were obtained from cDNA and gDNA, respectively. The deduced amino acid sequence from ORF had a high homology with DET3 from Arabidopsis, rice, as well as maize. Quantitative real-time PCR analysis showed that GhDET3 expressed ubiquitously in all detected tissues and organs. However, obvious differences were investigated between samples detected. The accumulation of GhDET3 mRNA reached the highest level in 12DPA fibers, in which stage fiber cell elongated rapidly. The lowest level was detected in 0DPA ovules (with fibers), in which stage fiber cell initiated from epidermis. The transcript accumulation of GhDET3 mRNA in ovules shared a similar variation with that in in fibers. In addition, in vitro ovule culture experiment demonstrated that applying exogenous 24-EBL treatment to 4DPA ovules (with fibers) was capable of increasing the expression level of GhDET3, while the accumulative of GhDET3 increased in transgenic FBP7∷GhDET2 cotton fibers in vivo. All these results pointed out that the GhDET3 gene played a crucial role in cotton fiber elongation.2. Gene function analysis of GhDET3 in transgenic tobaccoTo understand effects of GhDET3, a homologue of DET3 in cotton, p6-CaMV35S:.senseGhDET3 cassette was constructed and transformed into tobacco through Agrobacterium-mediated transformation. As a result, we found that the transgenic plants overexpressing GhDET3 promoted the vegetative growth. The plant height and leaf size were increased relative to wild-type, and the length of hypocotyl and root of transgenic seedling were longer than that of wild-type either in the light or dark. Moreover, overexpression of GhDET3 also resulted in the achene weight and seed size increased. Furthermore, transgenic seedling that compared with wild-type was more sensitive in response to gibberellin and 24-epibrassinolide. On the contrary, the transgenic seedling was insensitive in response to ABA and NaCl in the manner of seed germination and seedling elongation. Together with our data provided, the results indicated that GhDET3 plays a significant role in plant growth, while it was proposed that GhDET3 might be the most significant subunit that is responsible for BRs-induced cell elongation and acts as a master accounting for regulating stress tolerance.3. Gene function analysis of GhDET3 in transgenic cottonTo understand roles of GhDET3 gene in the development of cotton fiber growth and development, we constructed p6-CaMV35S∷antisenseGhDET3 vector and transformed into cotton plants, by means of Agrobacterium-mediated transformation. And then, the positive lines were screened and confirmed through both histochemical staining of GUS activity and PCR analysis of GUS gene. Comparing with wild-type, quantitative real-time PCR analysis indicated expression levels of GhDET3 in transgenic plants were inhibited dramatically. Suppression of GhDET3 expression also resulted in changes of phenotypes in transgenic cotton plants. Analysis of basic organs and tissues revealed that transgenic plants exhibited dwarf, reduced leaf size, shorten branches, and decreased apical advantages. Cotton boll produced from the antisenseGhDET3 plants were easy to abort between 4DPA to 6DPA. Thus, it is impossible to detect the quantity and quality of mature fiber. Based on the technologies of SEM and paraffin sections, 0DPA-2DPA ovules from wild-type and transgenic plants were analyzed. The results revealed that less fiber cells of 0DPA and 2DPA were detected in transgenic plants, comparing with wild-type, while the elongation of fibercell was also suppressed in transgenic plants. Furthermore, ovules from 0DPA balls were separatedand cultured in BT medium for 10 d in vitro, the resulted demonstrated GhDET3 gene play a crucialrole in fiber cell elongation.