Functional Analysis Of Cotton (Gossypium Hirsutum L.) Rac Protein Gene (GhRacA)

Cotton is the most important fiber crop in the world. Although great achievement has been obtained, it is very difficulty to improve the fiber productivity and quality by traditional breeding. Gene engineering provided a new strategy and broad perspectives to cotton breeding, but its application was blocked by lack of cloned genes key to initiation and elongation of cotton fiber. Therefore, it is very important to clone fiber-related genes and to elucidate their biological functions.Rac proteins, belonging to the subfamily of small GTPase, are an important group of molecular switchs on plasma membrane. Rac proteins are regard as hubs for signal integration and diversification in plant . Major target response systems known for Rac proteins include the actin cytoskeleton, membrane trafficking, reactive oxygen species production, hormone response and polarity establishment. Previous researches revealed that cotton Rac proteins participated in the secondary cell wall formation and regulated H₂O₂-mediated signaling.To investigate the fuctions of Rac proteins in fiber development and quality establishment, a Rac homologous gene (GhRacA) had been cloned from cotton fiber. Sequence and expression analysis showed that GhRacA encoded a group II Rac protein, and was expressed preferentially in developing fibers. In this thesis, a RNAi vector of GhRacA was contructed, the overexpressing and antisense, and RNAi vectors were transformed into cotton, and the phenotypic variations of transgenic cottons were analyzed.The main results were as follows:1. A RNAi construct of GhRacA was cloned into an expressing vector p5 downstream to a consultative promoter CaMV35S.2. Seven vectors were transformed into cotton via Agrobacterium-mediated method. Obtained transgenic cotton plants included 3 independent lines of 35S::GhRacA (4 plants), 2 lines of GFP:.GhRacA (4 plants), 2 p5 lines (3 plants), 2 antisense GhRacA lines (3 plants), 6 GhRacA RNAi lines (18 plants), 2 GH3::GhRacA lines (6 plants), and 6 antisense GH3::Gh RacA lines (10 plants). Quantitative RT-PCR analysis indicated that the gene was expressed at high levels in transgenic plants, while the expression of GhRacA was significantly suppressed in GhRacA RNAi cottons.3. Tissue dying indicated that overexpression of GFP::RacA promoted H₂O₂ accumulation in leaves.4. Fiber measurement showed that the fiber length of GhRacA transgenic cotton is shorter than that of control, while fiber length of antisense GhRacA transgenic cotton has no distinguishable variation compared to the control.5. Ovule culture revealed that, after 20d culture, fiber length of the ovules overexpressing GFPr.GhRacA was significantly shorter than those of antisense GhRacA ovules and the control.6. By histological observation, no significant varation in fiber initiation was found among the transgenic plants, but elongation of fiber initials was inhibited in the GFP::GhRacA overexpressing cottons.Taken togather, overexpression of GhRacA may inhibit the elongation of cotton fiber, suggesting that this gene plays an important role in fiber elongation. This thesis paved way to finally elucidate the relationship between GhRacA and fiber development.