Introduction Of Mant Constructs Into Cotton Genome

Mannosyl transferase(mant) is cloned from Arabidopsis Arabidopsis thianala. It is believed that the gene encoding Mannosyl transferase(mant) universally exists in the genomes of all eukaryotic and prokaryotic organisms. Its coding product Mannosyl transferase transfers mannose from GDT-mannose to dolichol monophosphate to form dolichol phosphate mannose (dol-p-man), which serves as a donor of mannosyl residues on the lumenal side of the endoplasmic reticulum leading to N-glycosylation, glycosyl phosphatidylinositol membrane anchoring, and O-mannosylation of proteins. Mannose also is the basic component of saliva acid and plant lectin. These two kinds of glycoproteins are parts of plant cell wall components and highly related to disease and insect resistances of plants. Therefore, abnormalities of mannose metabolisms certainly result in the changes of plant resistibility to virulent microorganisms and pests, and form barriers to plant normal developments. In this study, the coding sequence of the mant gene was applied to fuse with anther-specific promoter, tetravalent enhancer plus anther-specific promoter to build the constructs of fused genes and antisense RNA gene, and with which to transform apex segments of upland cotton via Agrobacterium mediated transformation, and to make them over expression, anther specific expression, or suppress the expression of the homologues counterparts in the cotton genome in the purpose of creating of new disease- and insect- resistant cotton varieties and transgenic male sterile lines.The main results in this study were as follows:1. The above-mentioned three mant constructs were successfully transferred to the cotton genome by Agrobacterium mediated transformation. Total 66 T₀ transgenic plants were obtained.2. The primers were designed according to the sequences of NPTâ…¡and mant gene and applied to PCR analyses of To transgenic plants. Percentage of the plants with positive signals in nptâ…¡ primer reaction is 59.1%, that of the plants in mant primer reaction is 50%, and the percentage of the plants showing both positive signals of nptll and mant primer reactions is 36.4%.3. The T₀ transgenic plants displayed positive signals in PCR reaction were further analyzed in Southern blotting. The results showed that whatever the genomic DNAs digested with restriction enzymes of KpnI or EcoRI both had the polymorphisms of restriction fragments while probed with the PCR product of nptll gene. But the size of the fragments only varied 9kb-25kb among the plants, indicating that the foreign geneshad integrated into the different sites of the host genome, and that the To plants originated from different transformed cells and had a lower degree of chimera.The hybridization results taking the PCR fragment of mant gene as probe showed that digestions of EcoRl presented internal fragments with anticipated sizes.4. Field characterization of TO transgenic plants did not show any significant difference from the check test plants in phenotypes, fertility and pollen grain development. It is possible that the transgenic mutants with desirable traits, such as insect- and disease-resistances and male sterility, could be identified and isolated from the Tl segregating populations.