CAJ | 학술논문

以豫杂一号泡桐为材料,通过对影响AFLP技术体系的各主要因素的研究,建立了适于泡桐AFLP分析的技术体系.结果表明最佳酶切体系(20μL)为500 ng模板DNA,3 U的Pst 1和Mse I,在37℃下双酶切3 h;20μL最佳连接体系中为酶切产物15 μL,0.25 μmol·L-1Pst I接头,2.5 μmol·L-1 Mse I接头,1 μL 10×T4 Buff-er,2 U T4连接酶,22℃连接18 h;20 μL最佳预扩反应体系中5 μL稀释10倍的连接产物,100 μmol·L-1dNTP,2 U Taq酶,250 μmol.L-1 Pst I和Mse I引物(P+AGT/M+AGT),2 μL 10×PCR Buffer.20 μL最佳选择性扩增反应体系中5 μL稀释20倍预扩增产物,100 μmol·L-1dNTP,2 U Taq酶,350 μmol·L-1 Pst I和Mse I引物(P+AGT/M+AGT),2 μL 10×PCR Buffer.最后,筛选出了97对适宜于泡桐AFLP分析的引物.
이예잡일호포동위재료,통과대영향AFLP기술체계적각주요인소적연구,건립료괄우포동AFLP분석적기술체계.결과표명최가매절체계(20μL)위500 ng모판DNA,3 U적Pst 1화Mse I,재37℃하쌍매절3 h;20μL최가련접체계중위매절산물15 μL,0.25 μmol·L-1Pst I접두,2.5 μmol·L-1 Mse I접두,1 μL 10×T4 Buff-er,2 U T4련접매,22℃련접18 h;20 μL최가예확반응체계중5 μL희석10배적련접산물,100 μmol·L-1dNTP,2 U Taq매,250 μmol.L-1 Pst I화Mse I인물(P+AGT/M+AGT),2 μL 10×PCR Buffer.20 μL최가선택성확증반응체계중5 μL희석20배예확증산물,100 μmol·L-1dNTP,2 U Taq매,350 μmol·L-1 Pst I화Mse I인물(P+AGT/M+AGT),2 μL 10×PCR Buffer.최후,사선출료97대괄의우포동AFLP분석적인물.
The establishment of Paulownia AFLP reaction system and its primer selection were investigated through study on the main factors affecting the system quality with Paulownia tomentosa × Paulownia fortune.The results indicated that the optimum conditions for restriction enzyme digestion of the genomic DNA from the Paulownia plant were as follows: 500 ng DNA template, 3 U Pst Ⅰ and Mse Ⅰrespectively in 20 μL reaction volume at 37 ℃ for 3 h;The ligation reaction (20 μL) with 15 μL the digestion product, 0.25 μmol·L-1 Pst I adaper, 2.5 μmol·L-1 Mse I adapter, 1 μL 10 ×T4 Buffer,2 U T4 ligation enzyme at 22 ℃ for 18 h was the optimum condition; the preamplification reaction (20 μL) with 5 μL the ligation fragment which was diluted 10 times, 100 μmol·L-1 dNTP,2 U Taq enzyme,250 μmol·L-1 Pst Ⅰ and Mse Ⅰ primer (P + AGT/M +AGT) ,2 μL 10 × PCR Buffer was the optimum condition; The selective amplification reaction (20 μL) with 5 μL preamplification product which was diluted 20 times, 100 μmol·L-1 dNTP,2 U Taq enzyme,350 μmol·L-1 Pst I and Mse Ⅰ primer (P + AGT/M + AGT) ,2 μL 10 × PCR Buffer.Finally,97 pairs of primers were chosen for the AFLP analysis of Paulownia plants.