Functional Analysis Of CesA9 And Expansin In Rice

Biomass is the major source of renewable carbon on the earth, and can provide enormous resource for large-scale economical production of transportation fuels and chemicals. Cellulose is the most abundant renewable biomass and is also major component of the plant cell walls. Understanding of cellulose biosynthesis is thus important scientifically. In this study, I characterized an important gene (OsCesA9) that involves in plant cellulose biosynthesis.1. Prepareration of cellulose synthase polyclonal antibodiesI prepared the antigens of OsCesA1, OsCes2, OsCesA3 with the specific peptide region, and the purified antigen were injected to rabbit for generating polyantibodies. Finally, all antibodies were detected by Western blotting analysis with specific bands and high activities.2. Phenotype observationThe phenotype of a T-DNA mutant was observed. It shows dwarfism and brittle clums, with the breaking force in leaf at 10% of the wild-type.3. Expression analysis of OsCesAsReal-time PCR was used to detect the expression level of OsCesAs. The results showed that the secondary cell wall CesAs (OsCesA9 and OsCesA4) transcript levels were significant decreased, whereas OsCesA7 had little change; OsCesA1, OsCesA3, OsCesA8, OsCesA2, OsCesA5, OsCesA6 appeared to have similar expression patterns to OsCesA9 and OsCesA4.4. Cell wall component analysisMutant (Oscesa9) had reduced crystalline and noncrystallin cellulose contents (47.6% and 30.15% of the wild type, and showed total hemicelluloses and pectins more that wild type by 8.08% and 23.89% respectively. The three main monomers of lignin (S, G, H) were reduced to some degree. 5. Fertility analysisCompared with the wild-type, the mutant Oscesa9 has some characterizations:the activity of pollen grain is lower; the anther is short and its color is light; the shape of some pollen grains are irregular.6. Compared with the wild-type, the activity of cellulose synthesis in vitro was significant decreased. Expansins are proteins that promote cell wall loosening and extension, which have a broad range of biological roles in diverse aspects of plant growth and developmemt, such as fruit softening, seed germination and floral organ development. Expansins are encoded by a superfamily of genes comprised of four subfamilies:a-expansins (EXPAs),β-expansins (EXPBs), expansin-like A (EXLA), and expansin-like B (EXLB) proteins. In this study, the relationship between Expansin and cell wall polysaccarides was analyzed.1. Microarray analysis of OsEXP familyA genome-wide microarray based gene expression analysis involving 33 stages of vegetative and reproductive development in two rice cultivars (ZS97 and MH63) revealed that the expression of OsEXP genes was tissue-specific. OsEXPA2, OsEXPA5, OsEXPA10, OsEXPA11, OsEXPA12, OsEXPA29, OsEXPBl, OsEXPB4,OsEXPB6 expressed signifcantly in the plumules and radicles of 2-day-old seedlings, also in the calli. OsEXPA9,OsEXPA12, OsEXPA13,OsEXPA14, OsEXPA17, OsEXPA22, OsEXPA23, OsEXPA24, OsEXPA28,OsEXPA31,OsEXPB5 expressed mainly in the radicles of 2-day-old seedlings and young roots. OsEXPA32, OsEXPA26, OsEXPB9, OsEXPBl a, OsEXPB10, OsEXLB1expressed specifically in the stamens and old panicles.2. OsExp/CslD/GT co-expression profilingExpression profiling analysis of ZS97 and MH63 revealed that some genes of OsExp/CslD/GT families co-expressed in the plumules (dark/light treatment), radicles (dark/light treatment) and stamens (one day before flowering).3. Co-expression analysis of OsExp/CslD/GT genes by RT-PCR and Real-time PCRSome co-expression patterens of OsExp/CslD/GT in ZH97 were verified by RT-PCR and Real-time PCR, which also verified in NPB and mutants (C1, C6, C15, G40).4. The relationship between Expansin and cell wallTo study the function of Expansins, we analyzed cell wall polysaccharides, the co-expression patterns between OsEXP and OsGT families, which may be involved in the hemicelluloses synthesis. In plumules, the results showed that OsEXPA1、OsEXPA10 may promoted the creep of xylan on the surface of cellulose. In radicles, through the analysis of cell wall polysaccharides, the co-expression patterns between OsEXP and OsCslD family, which may also be involved in the hemicelluloses synthesis, the results revealed that OsEXPB5 may promoted the creep of xylan on the surface of cellulose.In stamens, through the analysis of cell wall polysaccharides, the co-expression patterns between OsEXP and OsCslD family and the stamen-specific gene expression, preliminary data showed that OsEXPA26,OsEXPB9, OsEXPB1O, OsEXPB1a may promote the creep of pectin or hemicellulose on the surface of cellulose.