The Fiber Developmental Comparison Among Mutants And The Molecular Cloning And Functional Analysis Of Two Fiber Elongation Related Genes In Gossypium Hirsutum L.

Cotton is one of the most important economic crops in the world. Cotton fibers are derived from seed integument cells which undertaken a series of development and regulation. Thanks to the advantages with singled-cell structure and high content cellulose, it becomes one of the main materials for basic and applied researches in plants. Therefore, it is important to elucidate fiber development mechanism in the molecular level manner for theology and practice. In this study, dominant naked seed N1, recessive naked seed n2, Xuzhou-142 lintless-fuzzless (XZ142WX), Xinxiangxiaojilintless-fuzzless (XinWX), Xinxiangxiaojilinted-fuzzless (XinFLM) and super-short fiber mutant Lil were used for revealing the process of fiber initiation development via molecular biology and cytology methods, and cloning two important genes related to fiber elongation and further making clear their functions. These data facilitate the understanding of fiber development processes and provide the valuable gene resources for cotton transformation research in the future.1. Fiber initiation development study using different fiber-specific mutantsCotton fibers are single-celled trichomes derived from the outer integument of ovules. A greater understanding of the molecular mechanisms of cotton fiber development and regulation is needed so as to increase cotton yield, improve fiber quality and facilitate the development of novel man-made fibers. A series of fiber-specific mutants, or germplasms, have been recently utilized in the study of fiber development. In the current study, scanning electron microscope (SEM) was utilized to investigate developmental differences in lint and fuzz initiation in different genotypes(Gossypium hirsutum) of upland cotton. These fiber mutants included dominant naked seed Nl, recessive naked seed n2, Xuzhou-142 lintless-fuzzless(XZ142WX), Xinxiangxiaojilintless-fuzzless(XinWX), Xinxiangxiaojilinted-fuzzless (XinFLM), with TM-1, the cytogenetic and genetic experimental standard stock, as control. Characteristics of fiber initiation was analyzed-1, 0 and 1 days post anthesis (dpa) and at 4 and 5 dpa for fuzz initiation. Our data suggested that for TM-1, the lint initiation centered on day of anthesis(Odpa), and elongated significantly at ldpa, while fuzz initiation began at 4 dpa, although the shape of fuzz protrusions differed from that of lint fibers. Fiber initiation early occurred on the ovule funicular crest. Compared to control TM-1, there was a noted retardation in development and fiber protrusion hardly occurred in N1 and XinFLM. Additionally, the fiber protrusion density of the remaining five mutants was far lower on the day of anthesis. Microscopy data also demonstrated that two lintless-fuzzless mutants (XZ142WX and XinWX) developed protrusions during early developmental stages, although with irregular shape and unable to grow into fiber. Greater understanding of the differences in fiber development among these mutants will elucidate the developmental stages and the gene responsible for fiber and fuzz initiation.Based on the developmental retardation for the above mentioned mutants, we carried out RT-PCR using many fiber initiation related genes including GhE6、GhEXP1、 GhMYB1、GhMYB5、GhMYB6、GhMYB25、GhMYB36 and GhTTG1-GhTTG4 for N1 and XinFLM, n2 and TM-1 as controls. Results showed the expression of GhEXPl and GhMYB25 was lower in Nl and XinFLM than in TM-1 and n2, however, the expression of GhTTGl and GhTTG3 in XinFLM and n2 was higher than in TM-1 and N1. Further, in vivo treating with 30% H2O2, H2O and diluted 400-fold ethrel and in vitro culturing using acid BT(pH=3-4)medium and BT medium adding IAA (100μM) and GA3(100μM) for-1DPA ovules in XinFLM and N1 were performed, the results demonstrated that ovules treated with 30% H2O2 (hydrogen peroxide) induced fiber protrusions in XinFLM, while all treatments produced no evident effect on N1. To further confirm the relationship between gene expression and the effects of H2O2 in XinFLM, qRT-PCR analysis of four differentially expressed genes was performed using-1 day post anthesis (dpa) ovules of XinFLM treated for 24 and 48h with 30% H2O2 and H2O, with ODPA and 1DPA untreated ovules from XinFLM and TM-1 as control. The results showed that the gene expression of GhMYB25 and GhEXP1 showed significant difference in XinFLM after-1DPA ovule treated for 24 h relative to the untreated ovules and mock-treated ovules, with the gene expression of GhMYB25 increased significantly and that of GhEXPl reduced. This implied that H2O2 might be one of the upstream signal molecules affecting the expression of GhMYB25 and GhEXPl genes and fiber retardation initiation in XinFLM might be related with the amount of reactive oxygen species.2. The molecular cloning and function analysis of two fiber elongation related genesWD repeat genes play important roles in protein trafficking, RNA processing and signal transduction. In this study, we cloned and characterized a upland cotton WD40 repeat gene (GhWDR) by DDRT-PCR and RACE-PCR methods, and preliminarily analyzed its function by transformation and subcellular localization. Its transcripts widely distributed and were abundant during the fiber rapid elongation. Southern blot revealed the two copies in allotetraploid cotton. Localization study using a fusion protein consisting of the full length of GhWDR cDNA and GFP under the control of 35S promoter revealed that fluorescence was detected in both the cell wall and nucleus of onion epidemical cells and cotton stem epidemical cells. In addition, the transient expression experiment using cotton fiber at 6DPA, cotton leaf and Arabidopsis leaf indicated the green fluorescent signals mainly focused on the bases of fiber cells and trichomic cells. Like its expression in above mentioned cells, transgenic Arabidopsis plants were stained using GUS dye solution and the distribution of GUS protein also conferred the polarized characteristics. Interestingly, we detected their subcellular localization by constructing the different truncated constructs of this protein, founding out the importance of some amino acids for nuclear localization. Overexpression in fission yeast can significantly widen the yeast cells. Furthermore, this gene has correlation with cotton gland development and stoma on/off states and positively regulated by ABA hormone.Rab11 plays an important role in protein trafficking, especially new cell membrane and wall components transport to destined compartments or their recycling. In this article, a novel GhRABllc was isolated from the mutant and temporal-spatial expression pattern demonstrated that it probably played a negative role in cotton fiber elongation, i.e. no transcripts could be detected during the fiber initiation and elongation stages (-3-20DPA) and high expression occurred at the phage of secondary cell wall thickening (after 23DPA) in wild type, however, the much more early fiber development conferred abundant expressing (0-6DPA); southern blot revealed the single copy in allotetraploid cotton; subcellular localization assays showed its cell wall localization and polar distribution in the cell wall; both the stable and transient Arabidopsis transformation using GUS and GFP as reporter genes respectively indicated that this protein exclusively located the leaf trichomes; the resulting of in vitro ovule and fiber transforming and culturing showed that this protein inhibited the fiber normal growth; in vivo yeast over-expression was able to induce the yeast length significantly shorten; the 6DPA fiber and persimmon endosperm cells transient expression combining with the TEM experiments revealed that it participated in the Golgi vesicles trafficking; this gene located at the chromosome A8; In addition, DAB staining assay indicated that this protein might be involved in the H2O2 signal transduction. These results inferred that the fiber super-short phenotype of Li1li1 mutant resulted from the more early H2O2 production leading to enter into the cell wall thickening stage much earlierly.