Cloning, Expression And Subcellular Localization Analyses Of The Genes Encoding Cell Wall Proteins That Are Related To Fiber Development In Cotton (Gossypium Hirsutum)

Cotton Fibers are the seed hairs which originate from the epidermal cells of the ovular surface. Cotton fibers are not only the major natural fiber material for textile industry, and also as a unique system for studying cell elongation, secondary wall formation and cellulose synthesis. Fiber cells undergo four overlapping developmental stages: fiber cell initiation, elongation, secondary wall deposition, and maturation. In this study, four fiber-specific genes encoding cell wall proteins were isolated in cotton, and their expression profiling and subcellular localization were also analyzed. The main results are as follows:1. By using macroarray technique, twelve fiber-preferential genes encoding cell wall proteins, including four extensins, seven PRPs and one GRP, were isolated and characterized in cotton. Real time RT-PCR analysis revealed that three GhPRPs were specifically expressed in fiber. Two GhPRP, named as GhPRP3 and GhPRP5, were selected for further research. The sequence analysis showed that GhPRP3 contains an open reading frame (ORF) of 333bp which eneoded a polypeptide of 111 amino acids, and GhPRP5 has an open reading frame of 546bp which eneoded a polypeptide of 182 amino acids. GhPRP3 protein shares low homology with GhPRP5. Furthermore, both proteins also share very low similarity with the other plant PRPs. Thus, we speculate that the two proteins belong to new type PRPs. In addition, real time RT-PCR and Northern blot analyses revealed that expression of the two genes was regulated in cotton fiber development stages.2. To investigate the expression profiling as well as the roles of FLA genes during fiber development, nineteen GhFLA genes (cDNAs) were isolated from cotton (Gossypium hirsutum). Structural characteristics of the GhFLA proteins deduced from the isolated cDNAs were analyzed. The results showed that each of all 19 putative GhFLAs contained N-terminus signal peptide, one or two fasciclin-like domains and 1-3 AGP domains. Fifteen out of the nineteen FLA proteins are predicted to be glycosylphosphatidylinositol-anchored signal. The isolated cotton FLAs could be devided into four groups based on the domain organization, such as the number of fasciclin-like domains, AGP domains and the presence or absence of a putative GPI anchor signal. Real-time quantitative RT-PCR analysis indicated that the GhFLA genes are differentially expressed in cotton tissues. Three genes (GhFLA 1/2/4) were specifically or predominantly expressed in 10 DPA fibers, and the transcripts of the other four genes (GhFLA6/11/14/15) were accumulated at relatively high levels in cotton fibers. Highest levels of GhFLA5, GhFLA8 and GhFLA9 transcripts were detected in leaves, whereas the other cotton FLA genes were preferentially expressed in hypocotyls. Furthermore, expressions of the fiber-preferential GhFLA genes were regulated in fiber developmental stages and in response to phytohormones and NaCl. The identification of cotton FLAs will facilitate to study their roles in cotton fiber development and cell wall biogenesis.3. Promoters of the four fiber-specific genes, named as pGhPRP3, pGhPRP5, pGhFLA1 and pGhFLA4, were isolated using the method of genomic walking PCR. Many putative regulatory elements, suach as those related to light and low temperature response, phytohormone response, abiotic stress response, and so on (cis-elements), were found in the four promoters. Promoter::GUS fusion expression vectors were constructed and transformed to tobacco by stand leaf-disc method and to Arabidopsis by floral dip method, and the expression patterns of pGhFLA1 and pGhFLA4 were determined by GUS staining. The results showed that GUS gene driven by the pGhFLA1 promoter was expressed in the stem vascular tissues, vein and petiole of wounding leaves and epidermal cells of seeds in tobacco. Furthermore, GUS activity driven by the pGhFLA1 promoter was detected in the petiole trichome of some transgentic tobacco plants.4. GhFLAl::eGFP and GhFLA4::eGFP fusion expression vectors were constructed and introduced into cotton by agrobacterium-mediated method. The green fluorescence was detected in the cell wall of cotton callus cells using Confocal laser scanning microscopy. These results revealed that both GhFLA1 and GhFLA4 encode cell wall proteins.5. GhFLA1 and GhPRP3 overexpression and RNAi vectors were constructed and introduced into tobacco and cotton. The PCR results showed the heterologous genes were integrated into the genomes of 12 lines of GhPRP3 and 14 lines of GhFLA1 transgentic tobacco seedlings. The overexpression and RNAi vectors of GhPRP3 and GhFLA1 were transformed into cotton. Up to now, we obtained 5 positive transgenic cotton lines overexpressing GhFLA1. Further functional analyses are under way.