椭圆食粉螨ISSR-PCR反应体系正交优化试验设计  

Optimization for ISSR Reaction System of Aleuroglyphus ovatus with Orthogonal Design

作　　者：桂梓[1] 舒畅 钟玲 邹志文[1] 余丽萍[1] 夏斌[1] 

机构地区：[1]南昌大学生命科学学院,江西南昌330047 [2]江西省植保植检站,江西南昌330096

出　　处：《江西科学》2008年第4期622-625,共4页Jiangxi Science

基　　金：江西省自然科学基金(编号:2007GZN1925)"应用ISSR分子标记对不同群体肉食螨遗传多样性分析";江西省科技厅科技重大支撑项目"巴氏钝绥螨控制果蔬害螨技术研究与应用"。

摘　　要：以椭圆食粉螨基因组DNA为模板,采用正交试验设计方法,建立椭圆食粉螨的ISSR最佳反应体系。优化得到的反应体系为:Mg2+浓度1.0 mmol/L、Taq DNA聚合酶1.0 U、dNTPs浓度0.2μmol/L、引物浓度0.3μmol/L、模版DNA量40 ng。反应程序为:94℃预变性5 min;94℃变性1 min,48℃、50℃、52℃(不同引物退火温度各异)复性1 min,72℃延伸1.5 min,35个循环;72℃延伸5 min;4℃保存。通过梯度退火试验,确定不同引物的最适退火温度。Based on the genomic DNA of Aleuroglyphus ovatus, the factors influencing ISSR system were explored with the L16 （45） orthogonal design. The optimal conditions for ISSR reaction system （25 μl）were determined as follows： 1.0 U of Taq DNA polymerase, 1.0 mmol/L of Mg^2＋ ,0.2μmol/L of dNTPs mixture,40 ng of template DNA, and 0.3 μmol/L of each primers. The reaction program was as ： initial denaturation for 5 min at 94℃, 35 cycles of denaturation for 1 min at 94℃, annealing for 1 rain at 48℃ - 52℃, extension for 1.5 min at 72℃, with a final extension of 5 min at 72℃. The optimal annealing temperature for ISSR-PCR reaction was proposed by gradient PCR. Total of 100 primers subjected to three different annealing temoeratures.

关 键 词：椭圆食粉螨 ISSR 正交设计 反应体系 

分 类 号：Q5-33[生物学—生物化学]