4种楠木AFLP反应体系优化建立

Optimization of an AFLP protocol with four Phoebe species

采用核酸电泳缓冲液(TNE)结合十六烷基三甲基溴化铵(CTAB)提取的高质量楠木Phoebe基因组脱氧核糖核苷酸(DNA)可满足扩增片段长度多态性(AFLP)分析的要求。利用浙江楠Phoebe chekiangensis基因组DNA优化建立楠木AFLP反应体系如下:酶切反应300 ng基因组DNA,0.3μL缓冲液4,0.3μL牛血清白蛋白(100×BSA),50nkat EcoRΙ,25 nkat MseΙ,双蒸水加至30.0μL放置37℃恒温箱酶切4 h,聚合酶链式反应(PCR)仪上65℃15min;连接反应20.0μL酶切产物,2.5μL T4缓冲液,666.8 nkat T4连接酶,5 pmol EcoRΙ接头,10 pmol MseΙ接头,双蒸水加至25.0μL,16℃连接过夜(10～14 h);预扩增反应2.0μL连接产物,2.0μL 10×缓冲液,31.25nmol镁离子,16.67 nkat TaqDNA聚合酶,4 pmol脱氧核糖核苷三磷酸(dNTP),预扩增引物(E+A,M+C)各6 pmol,双蒸水加至20.0μL;选扩增反应稀释40倍的预扩产物2.0μL,2.0μL 10×缓冲液,31.25 nmol镁离子,4 pmol dNTP,16.67 nkat TaqDNA聚合酶,选扩增引物(M+CNN)15 pmol,选扩增引物(E+ANN)12 pmol,双蒸水加至20.0μL。利用上面体系在64对选扩引物中筛出13对适于浙江楠AFLP分析的最佳引物组合。

To meet the needs of an amplified-fragment-length-polymorphism （AFLP） protocol,high quality genome DNA was extracted from Phoebe chekiangensis,Phoebe sheareri,Phoebe zhennan,and Phoebe bournei seedlings with improved cetyltrimethylammonium bromide （CTAB）methods.For optimization of restriction system （30.0 μL）,the genome DNA,300 ng; was buffered with 0.3 μL; bovine serum albumin （BSA）,0.3 μL; EcoRI,50 nkat; and MseI,25 nkat; and incubated at 37 ℃ for 4 h; then the restriction enzyme was killed with 65 ℃ for 15 min.The best ligation system（25.0 μL） was with 20.0 μL of the digestion products; T4 buffer,2.5 μL; T4 ligase,666.8 nkat; EcoRI adapter,5 pmol; and MseI adapter,10 pmol;and left at 16 ℃ overnight （10-14 h）.The optimal preamplification system （20.0 μL） included 2.0 μL ligation products; 10 × buffer,2.0 μL; Mg2＋,31.25 nmol; Taq DNA polymerase; 16.67 nkat; dNTP,4 pmol; pre-amplification primers （E＋A）,6 pmol; and pre-amplification primers （M＋C）,6 pmol.Also,the best selective amplification system （20.0 μL） was 2.0 μL,1/40 pre-amplification products; 10 × buffer,2.0 μL; Mg2＋,31.25 nmol; dNTP,4 pmol; Taq DNA polymerase,16.67 nkat; amplification primers （M＋ CNN）,15 pmol; and amplification primers （E＋ANN）,12 pmol.Moreover,screening with the optimized AFLP reaction system resulted in 13 pairs of suitable amplification primers for AFLP analysis of Phoebe chekiangensis.