| Ammopiptanthus nanus(M.Pop.) Cheng f.comes from the Tertiary period and is the only broad-leaved evergreen shrubs at desert region in central Asia,it has been identified as a country secondary,endangered plant.Because of its evergreen and extreme tolerance of very cold and drought,especially fighting a low temperature(-30℃),the special chemical composition(antifreezing protein,AFP) has been researched,extracted and transfered as a most valuable material.The full-length open reading frame(ORF) of gene sequence from A.nanus AFP was cloned through RT-PCR and combined with RACE strategy.The results are as follows:1.As experimental material of A.nanus seedlings,the AFP gene full-length cDNA has been coloned by the cloning technology of RT-PCR and the strategy combined with RACE(Accession Number:GQ200581).The sequence length is 609bp,which encodes 203 amino acids.The congenetic analysis showed that their homology is only 65.7%on the AFP gene sequence compared with the same genus plant of A.mongolicus(Maxim) Cheng f.,and the encoding proteins homology of amino acid sequences is 76.68%.2.Extracting genomic DNA,then designing and and amplifying the full-length primer sequences of its ORF,the DNA sequencing result is same to full-length open reading frame sequence and the gene is continuous,without introns.At the same time it also shows that the gene in plants are highly conserved in evolution,possibly as an evolutionary molecular marker of plant research.3.A.nanus AFP secondary structure analysed by the molecular biology software.The results showed that most of AFP random coil is 72.9%.Meanwhileβ-sheet structure percentage(19.21%) is significantly higher thanα-Spiral(7.88%).These random coil andβ-sheet may be related with the performance of the antifreezing protein. |
