CAJ | 학술논문

[目的]为钩距虾脊兰种质资源研究奠定基础.[方法]以钩距虾脊兰为试验材料,利用改良[目的]CTAB 法提取钩距虾脊兰基因组 DNA,并对影响 ISSR 扩增反应的各因素进行优化.[结果]获得了高质量的钩距虾脊兰基因组 DNA;同时,建立了最适的钩距虾脊兰 ISSR-PCR 体系,即25 μl PCR 反应体积中,2.5 μl 10×PCR buffer,2.0 mmol/L MgCl_2,100 ng 模板 DNA,240 μmol/L dNTPs,1.75 U Taq DNA 聚合酶,0.4 μmol/L引物;最佳扩增程序为94 ℃预变性5 min,然后进行40个循环:94 ℃变性 30 s,复性温度根据各引物[目的]TM 值略低 1～2 ℃,30 s,72 ℃延伸50 s,循环结束后72 ℃延伸 7 min,4 ℃保存.[结论]这一优化系统的建立为进一步利用 ISSR 分子标记技术进行钩距虾脊兰遗传多样性研究提供了基础.
[목적]위구거하척란충질자원연구전정기출.[방법]이구거하척란위시험재료,이용개량[목적]CTAB 법제취구거하척란기인조 DNA,병대영향 ISSR 확증반응적각인소진행우화.[결과]획득료고질량적구거하척란기인조 DNA;동시,건립료최괄적구거하척란 ISSR-PCR 체계,즉25 μl PCR 반응체적중,2.5 μl 10×PCR buffer,2.0 mmol/L MgCl_2,100 ng 모판 DNA,240 μmol/L dNTPs,1.75 U Taq DNA 취합매,0.4 μmol/L인물;최가확증정서위94 ℃예변성5 min,연후진행40개순배:94 ℃변성 30 s,복성온도근거각인물[목적]TM 치략저 1～2 ℃,30 s,72 ℃연신50 s,순배결속후72 ℃연신 7 min,4 ℃보존.[결론]저일우화계통적건립위진일보이용 ISSR 분자표기기술진행구거하척란유전다양성연구제공료기출.
[ Objective ] The research aimed to lay the foundation for the germplasm resources researches of Calanthe graciliflora Hayata. [ Method] With C. graciliflora as the test materials,the genomic DNA was extracted from C. graciliflora by using improved CTAB method. Different factors that affected ISSR amplification reaction were optimized. [ Result] High-quality genomic DNA was obtained from C. graciliflora Hayata. And the reaction system and amplified procedure that were suitable for C. graciliflora Hayata were as follows:25μl amplification reactions system contained 2.5μl 10×PCR buffer,2.0 mmol/L MgCl_2,100 ng genomic DNA,240 μmol/L dNTPs,1.75 U Taq DNA polymerase and 0.4 μmol/L ISSR primer. The optimal amplified procedure was as follows: pre-denaturing for 5 min at 94℃ ,40 cycles of denaturing for 30 s at 94℃.annealing for 30 s due to 1 -2℃ lower than denaturing temperature of different primer,extending for 50 s at 72 ℃,finally extending for 7 min at 72℃. and presering at 4 ℃. [ Conclusion ] The optimal system could provide a basis for further study on genetic diversity of C. graciliflora Hayata by ISSR molecular marker.