外源DNA导入彩棉后RAPD分析体系的优化

通过25keV的10×1015Ar+/cm2离子介导将白棉全DNA导入彩棉进行RAPD分析,以改进CTAB法抽提叶片总DNA,进行随机扩增多态DNA(RAPD)分析,分别测试了镁离子、dNTP、模板DNA含量、引物浓度、DNA聚合酶量和牛血清蛋白浓度对反应结果的影响,通过各因子的组合研究,得出彩棉RAPD分析较适宜的扩增条件。应用构建的优化体系对40条引物进行筛选,选出引物S53、S176可使新彩棉8号品种引起特异性扩增,该随机扩增效果稳定、清晰,各材料间图谱差异明显,此试验奠定了利用RAPD技术对离子束介导外源DNA随机片段转化彩色棉品种进行鉴定分析的基础。

OPTIMIZATION OF RAPD CONDITIONS FROM EXOGENOUS DNA INTRODUCED INTO COLORED COTTON

The total DNA of white cotton was introduced into colored cotton by low energy beam Ar~+ ion with 10×10~(15) Ar~+/cm~2,25 keV.Random amplified polymorphic DNA(RAPD) was used to evaluate DNA variation of these varieties and to analyze each hereditary variation.It was necessary to determine the optimized system of RAPD reaction before analyzing the genetic diversity of colored cotton.The genomic DNA of colored cotton was extracted by the improved CTAB method and RAPD amplification was carried out under the optimal reaction conditions of the content of Mg~(2+),dNTP,DNA templates,primers,DNA polymerase and bovine serum protein.It was indicated that the species of the Xincai 8 green cotton produced the special amplification by using the S53 and S176 primers,and the effect of amplification was stable and the bar of electrophoresis was clear.It demonstrated that the RAPD technique was an effective method in the total DNA introduced into colored cotton by Ar~+ ion.