导入马齿苋DNA的烟草D4代株系的SRAP分析

SRAP Analysis of D4 Lines of Purslane DNA-introduced Tobacco

采用花粉管通道法将马齿苋DNA导入烟草,并经4次连续自交获得第4代材料(D4),选择其中32个株系进行SRAP纯度分析。结果表明:通过条件优化建立了烟草SRAP-PCR反应体系,即每10μL溶液中含有0.2mmol/LdNTP、20ng模板DNA、30nmol/L引物、0.5UTaq聚合酶和2mmol/LMgCl2;最佳复性温度和循环次数分别为53℃和35次;选用118对引物组合构建了烟草D4代的指纹图谱,其中不同引物组合产生的DNA片段数目在10～30之间,大小分布于100～700bp,且共扩增出1052条扩增产物,其中10条带表现出多态性,多态性比率仅为0.95%,说明使用SRAP标记检测烟草后代纯度是可行的。

By introducing purslane DNA into tobacco via pollen-tube pathway,the 4th generation self-bred progeny （ D4） was obtained,and the purity of 32 selected D4 were analyzed. The results showed that the optimum SRAP - PCR reaction system includes a solution of 10 μL,which contains 0. 2 mmol/L dNTP,20 ng template DNA,30 nmol/L primer,0. 5 U Taq polymerase and 2 mmol/L MgCl2,annealing temperature 53 ℃ and cycling 35 times. The fingerprint of D4 was established by combining 118 selective primers,the DNA segment number ranged from 10 to 30 with a size of 100 - 700 bp in different primer combinations,a total of 1052 bands were amplified and 10 of the bands expressed polymorphism,which accounted for only 0. 95% . It indicated that testing the purity of tobacco’s progeny with SRAP mark was feasible.