| Cotton (Gossypium spp.) is an important economic crop and the largest source of textile fiber in the world.Cotton fibers are unicellular trichomes origiating from the outer epidermal layer of ovules and regulated by numerous genes which ultimately affect the fiber quality and yield. So, it is important to elucidate fiber development process and major factors related to fiber quality by molecular biology and it also will provide important theoretical basis for molecular breeding of fiber quality. In this study, we had cloned a negative regulatory gene, which was homologous to AtCPC. Analysis of its characterization, function and regulatory mechanism were studied. In the regulatory pathway of fiber development, a bHLH gene was identified. Genome-wide classification and evolutionary anlysis of the bHLH family were done in G.raimondii. The main results were as follows:1. Functional identification and Regulatory mechanism analysis of GhCPCMYB transcription factors (TFs) are widely distributed in plants, and constitute one of the largest transcription factor families. TFs have a wide range of physiological functions involved in plant development and metabolism.In Arabidopsis, many TFs were cloned which were associated with the trichome development. CPC is a negative regulatory factors in trichome development and competitively binding to bHLH, which resulting inhibitting trichome development.Expression analysis of GhCPC were carried out between TM-1 and Mutants by Quantitative real time-PCR (Q-PCR) in ovules and fibers. The results showed that it had a significantly higher expression in mutans than TM-1 in 0,1,3 DPA. In in vitro cultured ovules, fiber development was inhibited by the CPC gene and promoted by gibberellins, while this hormone inhibited fiber growth under high CPC transcript accumulation.In GhCPC tangenic lines, over expression of GhCPC leads to significant decreases in fiber length. Scanning electron microscopy (SEM) of ovules of negative control plants showed normal differentiation and rapid emergence of fiber cells from the surface of the ovule at 0-DPA. By contrast, the surfaces of the ovules from the transgenic plants were smooth with no appearance of fiber initiation. But at 1 DPA, the CPC-overexpression fibers start to develop. These indicated that the fiber development was delayed by CPC overexpression, but not completely suppressed. When CPC was suppressed, no significant changees were observed between CPC antisense tangenic lines and negative control. This is similar to cpc mutant of Arabidopsis. We presumed that it was due to the presence of genes similar to AtETCl, AtETC2, AtTRY, which maked up the complementary function of GhCPC.Due to the high homology between GhCPC and AtCPC, it was suggested that GhCPC might share a similar regulatory pathway with AtCPC. Q-PCR analysis of the genes involed in fiber regulatory pathway showed that expression of GhHOX3 and GhRDL1 was inhibited by CPC overexpression. It was indicated that GhCPC regulated the downstream gene transcripts by a MYB-bHLH-TTG1 complex.In order to elucidate GhCPC regulatory mechanism for fiber development, in this study, the full length of GhCPC fused with the GAL4-DNA binding domain was used to screen the fiber Yeast Two Hybrid library. Co-transformed result showed that GhCPC had a strong interaction with a bHLH gene (denominated GhMYCl based on homology), while GhCPC also had an interaction with MYC1AC (bHLH-MYC_N domain) and MYC1ΔN (bHLH domain). Further study showed that GhMYCl could interact with GhTTGl and GhTTG4, but the interaction between them was not strong while no interactions were observed in GhTTG2 and GhTTG3. Together, these datas demonstrated that the existence of CPC-MYC1-TTG4 regulatory complex might play an important role in fiber development. As in Arabidopsis, it is a model of inhibition with feedback to explain the patterning mechanism of fiber in cotton.As the existence of regulatary complex, it plays a role in the regulation of downstream genes. In GhCPC transgenic plants, the transcripts of GhHOX3 and GhRDL1 were suppressed by CPC overexpression, suggesting GhCPC played its role in the form of GhCPC-GhMYC1-GhTTGl/4 complexes to regulate the downstream genes GhHOX3. Yeast one Hybrid was carried out in order to identify if GhMYC1 could bind to the E-box cis-element of GhHOX3 promoter. The result showed that GhMYC1 had the capability of binding to E-box, and activated the expression of AurR to obtain AbA resistant.In this stduy,we found that the regulatory model in cotton was not identical with Arabidopsis.In Arabidopsis, CPC overexpression caused the leaves with non-trichomes, so as in transgenic cotton with shoter fibers. Besides of the change in the fiber of transgenic cotton and mutants, no significant changes were found on leaf and stem trichomes. It also showed that there was no relationship with tissue-specific of GhCPC. So we hypothesized that the development of cotton fiber and leaf (or stem) trichome were regulated by two separate system and the regulatory complex was not suit to cotton fiber but leaf (or stem) trichome. Although the findings of this study is similar to Arabidopsis model, the development of the cotton fiber is much more complex than Arabidopsis trichomes. Differences between the them can further explain the molecular regulatory mechanism of plant trichome.GhMYC1 is an important component of the regulatory complex. In order to investigate the conservative and possible functions of other bHLH proteins in Craimondii, we constructed a Phylogenetic Tree of 177 GrbHLH and 127 AtbHLH proteins. The result of clustering and phylogenetic tree showed that it had a highly conservative of bHLH domain between these species. We also found that GhMYCl and AT5G41315.1 (GL3) had a proximate evolutionary relationship which could verified the function of GhMYC1. Therefore, the possible functions of bHLH proteins in G.raimondii could be speculated by evolutionary conservative and function of bHLH gene in Arabidopsis. All these will provide an evidence for further reserch. |
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