Characterization And Functional Analysis Of Cotton Fiber Expressed Protein Gene In Gossypium Hirsutum

Cotton (Gossypium spp.) is an important economic crop and the largest source of textile fiber in the world. Cotton fibers are highly elongated single-cell trichomes that arose from the outer epidermis of the ovules. As the single-cell structure without the complication of cell division and multicellular development, cotton fiber was considered to be a unique experimental system for investigating the regulation of fiber related genes. The improvement of the cotton transformation efficiency and isolation of fiber-specific genes provided a platform for production of transgenic cotton and function analysis of cotton key genes. There is great significance for elucidating the function of fiber-specific genes, as is enable to promote the understanding of cotton fiber development mechanism and improve cotton quality by means of molecular breeding.The gene with a 1274-bp 5’-complete cDNA and encoding a polypeptide of 331 amino acids, was isolated from a normalized cDNA library of upland cotton 7235 fiber, named GhCFE(Gossypium hirsutum cotton fiber expressed protein, Genbank No. DQ073045). The deduced protein sequence includes two domains of unknow function, DUF761 and DUF4408. Southern blotting of genomic DNA from upland tetraploid cotton showed that GhCFE is a two-copy-number gene in cotton. Genetic DNA sequences were cloned from diploid and tetraploid cotton (G.herbaceum var.africanum, G. raimondii, TM-1, H7124). Sequences alignment analysis conformed that GhCFE is conserved in different cotton species and contains two exons and one intron. Phylogenetic analysis revealed that GhCFE belongs to Dt subgenome, while CFE1 which has been reported before belongs to At subgenome.Bioinformatics analysis predicted that GhCFE contains a signal peptide which encoded by its N-terminal fragment (first 26 amino acid residues). To examine the subcellular localization of GhCFE, a green fluorescence protein (GFP) was fused with a linker to its C-terminus (GhCFE:GFP), under the control of the cauliflower mosaic virus (CaMV) 35S promoter, was transiently expressed in TM-1 protoplasts. Laser confocal imaging revealed that the GhCFE:GFP fusion protein was predominantly targeted to the plastid.The temporal-spatial expression analyses revealed that GhCFE transcripts were detected in the immature ovules of TM-1 as early as prior to the formation of fiber initials and reached the highest level in the 5DPA fiber. At the rapid elongation stage of cotton fiber, the expression was maintained moderate levels, yet the transcript abundance sharply decreased from 17 to 23DPA fibers. There was almost no expression in the roots and stems, additionally, in leaf, the expression of GhCFE was at a very low level. Dozens of times higher levels of GhCFE transcripts were commonly found in the 0,3 and 5DPA ovules of the fuzzless-lintless mutant (MD17, SL1-7-1 and Xu142 M) than fiber-bearing ovules of TM-1 in the same stage. These results indicate that GhCFE is highly expressed in cotton fibers and its up-regulated expression may not be conducive to lint and fuzz fiber initiation, and fiber elongation.Functional roles of GhCFE in cotton fiber development were analyzed using the anti-genetic technology. The sense-expression constructs of GhCFE driven by the E6 (E6S-SA) and the constitutive CaMV35S (35S-SA) promoter were developed using pBI121 as the fragment, respectively. Meanwhile, the antisense construct driven by the E6 (E6AS-SA) promoter was developed in the same means. Then, the plant-expressed vectors were transformed into the upland cotton WO genome by the Agrobacterium-mediated transformation. All of T0, T1, T2 and T3 transgenic plants were detected by the PCR analysis using the NPTII and promoter-gene specific primers. Finally,15 independant transgenic pure lines (including 4 35SC transgenic lines,5 E6SC transgenic lines,6 E6ASC transgenic lines) were obtained after three rounds selections. Southern blotting of genomic DNA from transgenic pure lines showed that most of the pure lines contain only one or two copies of transgene insertion of the NPTII. The expression level of GhCFE in ODPA (ovules with fibers), and 5DPA,10DPA,15DPA (fibers) of transgenic pure lines and WO was investigated by quantitative reverse transcription (qRT)-PCR. The accumulation of GhCFE transcripts was sharply increased in the ovules and fiber cells of the four 35SC transgenic pure lines compared with the wild-type plants(WO), especially the line of 159, in ODPA ovules the expression was twenty times more than W0, and in 5DPA fibers was ten times. Also, its expression in five E6SC transgenic pure lines was improved. However, the improvement level was lower than 35SC. Nevertheless, in 5DPA fibers of three E6ASC lines and 10DPA fibers of all lines, the expression of GhCFE was significantly reduced than that of the wild-type plants. Because of the overexpression of GhCFE, the fiber length of 35SC and E6SC transgenic lines was much shorter compared with WO. Yet, the fiber length of E6ASC transgenic lines was indistinguishable from the wild-type controls. In all of the 15 independant transgenic pure lines, there was no difference in fiber strengthen and fineness. Furthermore, scanning electron microscopy of the epidermal surface of randomly selected ovules showed a highly significant reduction of lint and fuzz initiation in the transgenic lines with overexpression of GhCFE than in WO in ODPA (the stage of lint initiation) and 4DPA (the stage of fuzz initiation). However, the ovules of the transgenic lines of E6ASC and WO were almost identical. Simultaneously, the overexpression of GhCFE proved by 35S promoter caused the obviously decreased number of stem trichomes.Identifying the proteins interacting with GhCFE would improve our understanding of the role of GhCFE during cotton fiber development. A fragment with ACTIN domain were identified by the full-length GhCFE fused with the GAL4-DNA-binding domain screening a yeast two-hybrid library, using the cDNAs from cotton ovules and fibers to construct prey vectors. Four GhACTs were recognized to interact with GhCFE after retransformed into the bait strains. These four genes were preferentially expressed in fiber. The bimolecular fluorescence complementation and transient gene expression systems verified the proteins interacted in plant. These results suggest that GhCFE interacts with GhACTs in living plant cells. Phalloidin staining noted that overexpression of GhCFE in transgenic cotton fibers promoting ACTIN transferred from cytoplasm to plastid, resulted less amount of and thinner actin bundles in 35SC and E6SC transgenic fibers, especially in 35SC transgenic fibers; whereas in E6ASC transgenic fibers, as the suppression level of GhCFE was no significant change, there were as much actin bundles as WO. Eventually, all reasons lead to shorter fibers in 35SC and E6SC transgenic lines, and no difference fibers in E6ASC.Additionally, in 35SC transgenic cotton and Arabidopsis, the expression levels of the transcription factors that positively regulate the fiber and leaf trichome initiation were generally declined. Speculated that GhCFE is upstream the complex of GL1-GL3/EGL3-TTG1 in the gene regulatory netwoks, and negatively regulates the start of cotton fiber.It is speculated that the over-expression of GhCFE affected the formation of "GL1-GL3/EGL3-TTG1" protein complex in the cotton fiber initial regulatory networks, thereby inhibiting the initiation of the cotton fibers, although, its mechanism should be further explored.