Functional Roles Analysis Of Cotton Pectate Lyase Gene And The Establishment Of Agrobacterium-Mediated Transformation System Of Embryogenic Calli In Cotton

Cotton fiber was operated by the genes expressed specifically during the fiber development. As a single cell without the complication of cell division and multicellular development, cotton fiber was considered to a unique experimental system for investigating the regulation of fiber-specific genes.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.In this study, functional roles of cotton pectate lysase gene (GhPEL) in cotton fiber development were analyzed using the anti-genetic technology. The temporal-spatial expression analyses revealed that GhPEL gene was fiber specific and preferentially expressed in fibers at 10 days-post anthesis (DPA). In addition, in vitro enzyme assays indicated that GhPEL exhibited cleavage-activity against de-esterified pectin. To further functional analysis, the antisense construct of GhPEL driven by the E6 promoter (E6ASP) were developed using pBI121 as the fragment. Then, this plant-expressed vector was transformed into the cotton genome by the Agrobacterium-mediated transformation. All of T0, T1 and T2 transgenic plants were detected by the PCR analysis using the NPTII and promoter-gene specific primers. We found that the transgenic lines containing the antisense GhPEL demonstrated the various changes in the fiber lengthen and less or no difference in fiber strengthen and fineness. Subsequently, six pure lines with one or two copies transgene insertion tested by southern blot analysis of the NPTII were selected to further analyze. The GhPEL expression during fiber elongating in the six transgenic cotton lines were significantly suppressed with the degree of inhibition varied. Further, the reduction of GhPEL enzymatic activity by decreasing GhPEL transcripts severely affected the degradation of de-esterified pectin in primary cell walls (PCW) of transgenic cotton fibers, which consequently blocked cell wall loosen in early fiber development. Ultimately, the fiber elongation of all these transgenic lines was repressed and showed the short-fiber phenotype. Integrating these results, the GhPEL may play an important role in the process of normal fiber elongation in cotton. This is also the first report about the functional role analysis of GhPEL in fiber development.In addition, GhPEL expressed specifically at the tetrad period. A sense-expression construct of GhPEL driven by the 35S promoter (35SP) was introduced into the WO, and the pollen of TO transgenic plants showed various degrees of abnormality and male sterile, this probably resulted from the overexpression of GhPEL driven by 35S promoter in pollen development. Alos, we found and selected two mutants whose phenotypes could be stably inherited into the next generation:a cotton curled leaf mutant and a dwarf mutant. These cotton mutants were purified, which could be the valuable materials using for cotton biology analysis.In addition, Agrobacterium tumefaciens mediated transformation of embryogenic calli (EC) with the higher efficiency was enhanced by choosing appropriate EC and improving efficiency of co-culture, selection cultivation and plant regeneration. We found that transgenes were inactived during the transformation and studied the mechanism of inactivation. Ninety-seven transgenic plantlets were identified by PCR amplification for GUS reporter gene and NPTâ…¡selection marker gene. Among the 97 transgenic plantlets,10 (about 10%) GUS inactive individuals were detected. After grafting the transgenic plantlets in the greenhouse, only one GUS-negative plant survived.In an effort to study the silencing mechanism of the GUS-negative transgenic plant, a GUS-positive plant generated from a single calli line was chosen for comparisons. Southern blot analysis revealed that the two transgenic plants possessed the same insertion copy numbers, and they had the same transformation event. GUS assay and RT-PCR analysis indicated that GUS was silenced in the GUS-negative plant but it was expressed in the GUS-positive plant; while RT-RCR detection showed that the NPTâ…¡gene expressed in both transgenic plants.Restriction endonuclease-PCR analysis of methylation in the 35S promoter was conducted by using of the methylation-sensitive enzymes Hapâ…¡/Mspl. The results demonstrated that the Hapâ…¡site in the TATA box of the 35S promoter region was methylated in the GUS inactive transgenic plant and not in the GUS active plant, which indicated that methylation of the 35S promoter caused GUS silencing in transgenic cotton plants.