珍稀植物杨叶肖槿ISSR体系建立及检测  

Detection and Establishment of ISSR System for Rare Species Thespesia populnea

作　　者：张永夏[1] 刘晓[1] 黎科[1] 关于元[1] 胡学强 邹永东[1] 

机构地区：[1]深圳大学生命科学学院,广东深圳518060 [2]深圳市绿化管理处,广东深圳518029

出　　处：《西北植物学报》2012年第4期829-834,共6页Acta Botanica Boreali-Occidentalia Sinica

基　　金：国家水体污染控制与治理科技重大专项(2008ZX07526);广东省科技项目([2009]198);深圳市科技项目(SY200806270107A);深圳市城管局项目(OTC1018212;201009;2010015)

摘　　要：针对珍稀植物杨叶肖槿ISSR反应的特点,建立了适用于杨叶肖槿遗传多样性研究的ISSR最适反应体系,具体包括:2.0μL 10×Buffer,27.5ng的模板DNA,2.0μL的dNTP,1U的Pyrobest DNA酶,1.25μmol/L的引物;最佳反应程序为94℃预变性5min,然后94℃变性1min,49℃退火45s,72℃延伸1min,35个循环;最后72℃延伸10min,4℃终止反应。应用该优化的反应体系筛选出了10条稳定性强、清晰度高而且表现出一定多态性的ISSR引物,并对杨叶肖槿进行了检测,获得了清晰稳定的扩增图谱。To seek a standardizing program of ISSR technique for genetic diversity analysis of Thespesia populnea ,a single factor experiment was designed to optimize ISSR PCR amplification system. The suitable reaction system was obtained,that is 20uL reaction system containing 2.0 uL of 10XBuffer,27.5 ng of to- tal DNA, 2.0 uL of dNTP , 1 U Pyrobest DNA polymerase and 1.25 umol/L of ISSR primer. The profile of ISSR-PCR was an initial denaturation step for 5 min at 94°C ,follow by 35 cycles of 1 min at 94°C ,45 s at annealing temperature 49°C ,1 min at 72°C ,and a final elongation 10 min at 72°C for one cycle,then termi- nation reaction at 4°C . Ten polymorphic primers were screened using this reaction system. Stable and clear amplification patterns were obtained, indicating the ISSR-PCR amplification system was feasible. The fac- tors affecting the amplification of genome DNA of Thespesia populnea were also discussed in the paper. It provides the basis on studies of germplasm resources and genetic diversity of T. populnea.

关 键 词：杨叶肖槿 ISSR PCR反应体系 优化 

分 类 号：Q789[生物学—分子生物学]