Cloning And Function Analysis Of A Fasciclin-like Arabinogalactan Protein Gene(GhFLA) From Cotton (Gossypium Hirsutum L.)

Plant cell walls are primarily composed of the polysaccharides cellulose, crosslinking glycans, pectins and some proteins. Most cell wall proteins belong to large families that include enzymes, expansins, wall-associated kinases and hydroxyproline(Hyp)-rich glycoproteins(HRGPs). Arabinogalactan proteins (AGPs) are a class of HRGPs. In Arabidopsis, the glycosylphosphatidylinositol(GPI)-anchored AGPs can be divided into four subclasses, The fasciclin-like AGPs (FLAs) constitute a fourth distinct subclass of AGPs.Previous studies have shown that AGPs may take part in plant cell enlarge in two fundamentally different ways including diffuse cell expansion and tip growth. In order to investigate the function of a cotton gene GhFLA, the coding sequence was placed under the CaMV 35S promoter, and introduced into tobacco and cotton.The main results are as follows:1. Cloning and characterization of GhFLAA cotton FLA (fasciclin-like arabinogalactan protein) gene, named GhFLA, was stochastically cloned from the cotton fiber cDNA pool. Sequencing showed that GhFLA cDNA was 1485 bp in length, containing a 291 bp 3'-UTR and an ORF of 1194 bp encoding a putative polypeptide of 397 amino acid residues with a predicted molecular mass of 42.8 kDa. Blast analysis showed that the predicted GhFLA protein contained fasciclin-like domains and AGP-like domains, and had 62%, 53% identity with the AtFLA2 and the putative rice endosperm specific protein SC3, respectively. A splicing signal peptide sequence and a precursor protein splicing sequence were found at the N-terminal and C-terminal of GhFLA, respectively. The expression of GhFLA was not observed in young stems, leaves and buds. However, its transcripts accumulated abundantly in root, ovules and fibers. The expression of GhFLA reached maximum at 12 DPA and ended at 18 DPA of fibers. 2. The elongation of root and fibers was inhibited by AGPs-specific inhibitor Yariv reagentIncreased expression of GhFLA in root and fibers suggests that FLAs may actually promoteroot and fiber cell elongation. We thus tested the effect of AGPs-specific inhibitor Yariv reagent on root and fiber cell growth by treating in vitro-cultured seeds and ovules with Yariv reagent. When 10 or 30μmol/L of Yariv reagent was added into the culture medium, the rate of root elongation decreased 42% or 46%, respectively. When cultured in BT medium with 10μmol/L Yariv reagent for 14 d, the fiber length of 0 DPA ovules reduced significantly. Addition of Yariv did not affect the final size of the cultured ovules, suggesting that the treatment did not have toxic effect and that AGPs may play a specific and important role in promoting fiber cell elongation.3. Overexpression of GhFLA accelerates seeds germination in tobaccoIn order to investigate the function of GhFLA, the coding sequence of GhFLA was placed under the cauliflower mosaic virus 35S promoter, and introduced into tobacco via agrobacterium tumefaciens-mediated transformation. The seed germination rate of transgenic tobacco was higher than that of control, and the higher germination percentage showed positive correlation with the high expression level of GhFLA, suggesting that GhFLA may accelerate seeds germination in tobacco.4. The effect of overexpression of GhFLA on fiber length, lint percentage and root elongationAs AGPs may have an important role in root and fiber development, we thus tested whether theoverexpression of GhFLA in cotton can also promote root or fiber cell elongation. The fiber of cotton overexpressing GhFLA was 25.5～27.4 mm in length, and the fiber length of untransformed cotton was 25.5 mm. No significant effect on lint percentage was observed between transgenic cotton and control. Compared with 22.6 mm of control, the root growth rate of T₁ transgenic cotton decreased 3.3 mm in FLA5-1 and 3.9 mm in FLA5-2 per 24 hr, but FLA3-1 increased 1.4 mm, Suggesting that the GhFLA may have no effect on fiber length, lint percentage of T₀ transgenic cotton and root elongation of T₁ transgenic cotton.