摘要
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.
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.
出处
《林业科学》
CAS
CSCD
北大核心
2000年第1期121-124,共4页
Scientia Silvae Sinicae
基金
国家"九五"攻关课题!(960110301)
关键词
杉木
RDNA
亲缘关系
克隆
序列测定
分类
染色体
Chinese fir (Cunninghamia Lanceolata Hook), rDNA,Internal transcribed spacer1(ITS-1), Relationship analysis, Cloning and sequencing
