The Establishment Of The Ryegrass Biolistics Transformation System And Salt Tolerance Gene Fragments Cloned

Effects of different concentration of 2,4-D on embryonic callus induction of perennial ryegrass was analyzed on MS medium. Transgenic plants had been regenerated from embryonic calli after bombardment with rice basic chitinase gene by microprojectile. Embryonic calli co-transformed with plasmid pARN6 (containing rice basic chitinase gene) and plasmid pD(containing gusA gene and bar gene) were selected and differentiated on medium supplemented with 2mg/l PPT. Regenerants were analyzed by Southern blotting and PCR. It has shown: (1)MS containing 5.0mg/L 2,4-D is optimal for embryonic callus induction. (2)Up to 243 plants were regenerated, and 18 plants containing RC24 gene, 15 plants containing bar gene, two plants harbouring both RC24 gene and bar gene were identified from them. (3) Using primers of RC24 gene, a 565bp fragment was amplified from ryegrass genome, it was analysed after sequencing.If amino acid sequences or nucleotide sequences of members in a same gene family are known, conserved domain can be found with bioinformatics soft, and degenerate primers can be designed to amplify homologous DNA fragments from other species. Full-length gene sequence can be cloned by RACE technology.Amino acid sequences or nucleotide sequences of Na+/H+ antiporter gene of six species were searched out from GenBank. 8 amino acid conservative regions and 7 nucleotide conservative regions were found after homologous comparison via DNASTAR 9.0. These regions were used to design degenerate primers.Plants of ryegrass, tall fescue, blue grass, bentgrass and shepherd's purse were treated with 200mmol/L NaCl for 12 hours. DNA and RNA were extracted from leaves.Using primer Natha and Nathb, a 530bp fragment was amplified from ryegrass genomic DNA and a 520bp fragment was amplified from bentgrass genomic DNA. After cloning and sequencing, DNA sequences were analysed in Genbank and no homologous sequences were found. However, a deduced amino acid sequence aftersix-frame translation have 54% similarity with rice bacterial blight-resistance protein Xal.Two fragments about 800bp were amplified from ryegrass genomic DNA and bentgrass genomic DNA with primer Nath2 and Complement Nath7. These two fragments have high similarity with Na+/H+ antiporter genes of other species. Deduced amino acid sequences also have high similarity with Na+/H+ antiporter amino acid sequences of other species, and the highest similarity reached 80%.