| Cotton fibers are single-celled seed trichomes that originate from the outer integuments of the ovule and undergo four distinct but overlapping developmental steps including initiation, elongation, secondary wall deposition, and maturation. During this process, fiber initials elongate by a factor of1000to3000. It is well-known that cytoskeleton rearrangement and dynamic change provide framework support, substance transport and signal transduction for fast cell expansion. Therefore, cytoskeleton is one of the most important factors involved in cotton fiber development.Ligon lintless-1(Li-1) is a shortened lint fiber mutant, the fiber cells degenerate and stop elongating early, which provide an ideal model for studying cotton fiber elongation mechanism. In our previous study, a differential proteome analysis was performed between the fibers of4and8days postanthesis (DPA) from Li-1and its isogenetic wild type of TM-1(WT), a protein was found to be down-regulated in Li-1, and thus it is proposed to be related to fiber elongation. This protein was further identified as villin, an actin binding protein, By using mass spectrometry analysis and database searching, which is most similar to the sequence of an EST (gi|109859616). On the basis of these results, the present study has been conducted and the main results were summarized as follow:1. According to this EST sequence, an actin binding protein of villin gene was molecularly cloned from cotton by RACE technology, and designated as GhVLNl for the moment. The open reading frame is2886bp, encoding a961-amino-acid protein with the theretical molecular weight of23.72kDa, and pI of4.87. BLAST analysis indicated that it has a high identity with other plant villin genes.2. The expression pattern of GhVLN1was characterized by real-time fluorescence quantitative PCR assay, and the results showed that this gene is predominantly expressed in roots, stems and fibers; and that at6DPA, the gene expression level in Li-1fiber is much lower than that in WT, suggesting the decreased expression of this gene is probably associated with the blocked fiber elongation in Li-1. 3. Subcellular location study indicates that the GhVLN1fusion protein is distributed in a fibriform network in cytoplasm, as this protein is functionally predicted to be a villin related to cytoskeleton, this experiment confirms gene funtion prediction result.4. The GhVLN1was integrated into a yeast expression vector (pREP-5N), and electro-transformed into fisson yeast. Morphologic changes were observed under fluorescence microscope with the fluorescence staining of DAPI. The results show that GhVLNl leads to a significant increase of length and length/width ratio of yeast, suggesting that ectopic expression of this gene influence the cytoskeleton structure and lead to polar expansion in yeast.5. Taking the wild type Arabidopsis as control, we evaluated the phenotypic features of five villin gene knockout lines, including AtVLN1, AtVLN2, AtVLN3, AtVLN4, AtVLN5. We found the heart leaves of AtVLN3and AtVLN5knockout lines appear yellow, the number of trichomes and root hairs decreased obviously, and the growth vigor was weak. These results indicate that loss of villin protein may lead to disturbed chlorophyll synthesis, reduced number of sites for trichome and root hair initiation, and abnormal seeding growth. However, not all of the villin gene knockout mutants showed phenotypic changes in seedling, suggesting that different villin genes have overlapped or complementary functions. |
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