CAJ | 학술논문

[目的] 优化适合于瘤胃甲烷生成菌的PCR反应体系.[方法] 以1.5周岁健康的藏系绵羯羊瘤胃内容物总DNA为模板,用特异性引物对甲烷生成菌16S rDNA保守序列进行PCR扩增,并通过改变PCR反应体系中的各参数,优化其PCR反应体系中的相关参数.[结果] PCR最优程序为:94 ℃预变性3 min;94 ℃变性45 s,51 ℃退火45 s,72 ℃延伸90 s,35个循环;72 ℃后延伸7 min.优化后的PCR反应体系为:5.00 μl 缓冲液,4.00 μl d-NTP,2.00 μl引物(前引物和后引物各1.00 μl),4.00 μl MgCl2,0.25 μl Taq DNA 聚合酶和1.00 μl 1∶ 100倍稀释的DNA模板,然后加水补足25.00 μl.[结论] 该研究为瘤胃甲烷生成菌功能的研究奠定了基础.
[목적] 우화괄합우류위갑완생성균적PCR반응체계.[방법] 이1.5주세건강적장계면갈양류위내용물총DNA위모판,용특이성인물대갑완생성균16S rDNA보수서렬진행PCR확증,병통과개변PCR반응체계중적각삼수,우화기PCR반응체계중적상관삼수.[결과] PCR최우정서위:94 ℃예변성3 min;94 ℃변성45 s,51 ℃퇴화45 s,72 ℃연신90 s,35개순배;72 ℃후연신7 min.우화후적PCR반응체계위:5.00 μl 완충액,4.00 μl d-NTP,2.00 μl인물(전인물화후인물각1.00 μl),4.00 μl MgCl2,0.25 μl Taq DNA 취합매화1.00 μl 1∶ 100배희석적DNA모판,연후가수보족25.00 μl.[결론] 해연구위류위갑완생성균공능적연구전정료기출.
[Objective] The aim was to optimize PCR reaction system suitable for methane producing bacteria in rumen.[Method] With the total DNA in rumen content of 1.5-year old healthy Tibetan sheep as template, the conservative sequence of methane producing bacteria 16S rDNA was amplified by PCR using specific primers.The related parameters in the PCR reaction system were optimized through changing each parameter in the PCR reaction system.[Result] The optimum PCR program was as follows: pre-denaturing under 94 ℃ for 3 min, then denaturing under 94 ℃ for 45 s, annealing under 51 ℃ for 45 s and extending under 72 ℃ for 90 s.After 35 cycles, the sample was extended under 72 ℃ for 7 min.The optimized PCR reaction system was as follows: 5.00 μl buffer, 4.00 μl d-NTP, 2.00 μl primers (upstream primer and down primer were 1.00 μl each), 4.00 μl MgCl2, 0.25 μl Taq DNA polymerase, 1.00 μl DNA (1∶ 100), then adding ddH2O to 25.00 μl.[Conclusion] The research laid the foundation for study on the function of methane producing bacteria in rumen.