珍稀濒危植物长序榆的ISSR-PCR引物筛选及反应条件的优化  

Establishment and optimization of ISSR-PCR amplification system in Ulmus elongata

作　　者：李文巧[1,2] 徐根娣[1,2] 吴玉环[3] 邱志军[1,2] 高建国[1,2] 刘鹏[1,2] 

机构地区：[1]浙江师范大学生态研究所,金华321004 [2]浙江师范大学植物学实验室,金华321004 [3]杭州师范大学生命与环境科学学院,杭州310036

出　　处：《安徽农业大学学报》2012年第4期560-567,共8页Journal of Anhui Agricultural University

基　　金：国家自然科学基金(30970188和30770161);浙江省自然科学基金(Y5090339)共同资助

摘　　要：采用正交设计试验,利用引物UBC834对长序榆ISSR-PCR扩增的主要影响因子Buffer(含Mg2+)浓度、模板DNA、引物浓度、Taq DNA聚合酶浓度、dNTPs浓度进行优化分析,并在此基础上对PCR反应过程中的退火温度、循环次数、延伸时间进行检测。结果表明,在25μL ISSR-PCR反应体系中各因素的最佳浓度为2.5μLBuffer(含Mg2+),50 ng模板DNA,2.5 U Taq DNA聚合酶,1.2μmol.L-1引物,0.2 mmol.L-1dNTPs,引物UBC834的最佳退火温度为57℃,最佳循环次数和延伸时间分别为35个循环和1 min。此外,还利用优化的反应体系成功筛选出11条ISSR引物,并用筛选的引物对长序榆的遗传多样性进行了分析,结果发现,在物种水平上,长序榆的遗传多样性较高,多态位点百分率(PPB)为82.35%,Shannon信息指数(I)为0.395 9,Nei’s基因多样性指数(H)为0.258 5。而种群水平上的遗传多样性则较低,多态位点百分率(PPB)、Shannon信息指数(I)、Nei’s基因多样性指数(H)分别为29.07%、0.173 3和0.119 1。To optimize the ISSR-PCR reaction condition for Ulmus elongata, the concentration of buffer, template DNA, primers, Taq DNA polymerase and deoxy-ribonucleoside triphosphate （dNTPs） were studied and analyzed with the orthogonal design experiment using UBC834 as primer. Then, based on the optimal ISSR-PCR amplification system, the annealing temperature, the number of cycles and the extension time were determined by gradient PCR. The results showed that a suitable ISSR-PCR amplification system for Ulmus elongata was estab- lished. In a total volume of 25 ~tL ISSR-PCR amplification system, 2.5 gL buffer, 50 ng template DNA, 2.5 U Taq DNA polymerase, 1.2 pmol·L^-1 primer and 0,2 mmol·L^-1 dNTPs were optimum. The optimal condition for primer UBC 834 was annealing at 57℃, 35 cycles, and extension 60 s. Eleven ISSR primers were screened by using the optimal reaction system, and the genetic diversity was analyzed with these primers. The results showed that at species level, the value of the average percentage of polymorphic bands （PPB） was 82.35%, Nei＇s genetic diver- sity index （H） was 0.258 5 and Shannon＇s information index （I） was 0.395 9. While at population level, the values ofPPB, H and 1were 29.07%, 0.173 3 and 0.119 1, respectively.

关 键 词：长序榆 ISSR-PCR 反应体系 优化 

分 类 号：Q946.3[生物学—植物学]