Cloning Of Cotton SGT Familly Genes And Antibody Prepration Of The Proteins Associated With Cellulose Synthesis

Cellulose is the most abundant biomass on the earth and takes up 50% of carbon in plant kingdom. Cellulose is the major component of plant cell walls. Cotton fibers consist of single cells, and have 95% cellulose. Therefore, cotton fibers become a model plant for investigating cellulose biosynthesis. Although cellulose synthase genes (CesAs) in cotton fibers have been identified, the mechanism of cellulose synthesis is still not very clear. Previous works reported that UDP-glucose:sterol glycosyltransferases (SGTs) synthesis SG, which may act as a primer for cellulose synthesis. Based on the 3 EST sequences of cotton fibers and the homologous sequences of other species, we cloned two sterol glucosyltransferase genes, and purified one recombinant protein for antibody preparation.It have been reported that endo-β-1,4-glucanase、β-1,3-glucanase and callose synthas were involved in cellulose synthesis. For further characterization of those proteins, we used prokaryotic expression system to purify these proteins, and then prepared their antibodies. The main results described below:1. The cDNA sequences of GhSGTl and GhSGT2 were cloned, the CDS of GhSGTl is 1878bp, encoded a non-transmembrane protein, the MW is 68690.67 Da, the CDS of GhSGT2 is 1983bp. encoded a transmembrane protein, the MW is 73350.45 Da, both GhSGT1 and GhSGT2 contain a activate site of glycosyltransferase.2. The genomic sequences of GhSGTl and GhSGT2 were obtained, the length of GhSGTl is 9186 bp, including 14 exon and 13 intron, the length of GhSGT2 is 7613 bp, including 15 exon and 14 intron.3. Semiquantitative RT-PCR results show that GhSGTl and GhSGT2 were strongly expressed at 4 DP A and 9 DP A of upland cotton,9 DPA and 14 DPA of sea island cotton, but the expression levels of other periods were relatively weak. The expression level of GhSGT2 is much lower than GhSGTl, and the expression patterns of GhSGTl and GhSGT2 are consistent with SuSy expression.4. Using prokaryotic expression system, we purified proteins of GhSGTl, endo-β-1,4-glucanase,β-1,3-glucanase and callose synthase for antibody preparation, and then we checked the quantity of antibodys with western blotting.