杉木rDNAITS- 1 区的克隆及序列测定

The rDNA internal transcribed spacer 1(ITS-1) regions of two Chinese fir provanences was amplified and cloned by PCR reaction.The PCR reaction was following:97℃ 5 minutes→95℃ 5 minhtes→Adding the Tag polymerase→94℃ l?min 56℃ 1?min,72℃ 2?min;thirty\|six cycles→72 ℃ 10?min.High quality DNA template is necessary for the amplification of ITS-1 sequence,during the PCR reaction,ten minutes denaturation time and 56℃ annealing temperature are beneficial to amplification.The ITS-1 fragment was ligated to PUC19 plasmid,digested with Hind Ⅱ and transformed into competence cells of E.coli JM83 strain,the cloned strains harboring recombinant plasmid were obtained,those recombinant plasmids were used to sequence for ITS-1 fragment.Sequence analysis indicated that the sequence length is 273 bp,the using percentage of A\,T\,C\,G within ITS1 sequence of Chinese fir were 27.5%\,23%\,21.6%\,27.9% respectively and the G/C content of ITS1 sequence was 48.35%.Comparing with other plants,the G/C content of Chinese fir was less than other plants,whose ITS1 regions have been sequenced.As to ITSI sequence,there was no difference among two Chinese fir provenances,sequence analysis disclosed there were two repeat sequences [AAAG] n and [TTG] nappeared within ITS1 sequence of Chinese fir.