我省板栗主栽品种(无性系)RAPD反应体系的建立

Establishment of RAPD Reaction System in Main Cultivation Varieties of Castanea mollissima in Zhejiang Province

在ＲＡＰＤ反应中，有许多影响结果的稳定性和准确性。本项研究采用浙江省８个板栗主栽品种（无性 系），对ＲＡＰＤ各种反应条件进行了探索，结果表明，板栗理想的反应体系为：２０μｌ反应体积含有１００ ｍｍｏｌ／Ｌ Ｔｒｉｓ－ＨＣｌ，５００ ｍｍｏｌ／Ｌ ＫＣＬ， ２０ｍｍｏｌ／Ｌ ＭｇＣＬ２， ０．０１％Ｇｅｌａｔｉｎ，ｄＡＴＰ、 ｄＴＴＰ、 ｄＣＴＰ、 ｄＧＴＰ的量各为 １００μｍｏｌ／Ｌ， ５０－２００ｎｇ引物， ０．７５－１．０单位Ｔａｑ酶， ２－１０ｎｇ模板ＤＮＡ。反应条件为： ９４℃预变性２ｍｉｎ， 再９４℃ ３０ｓ、４０℃３０ｓ、７２℃１．５ｍｉｎ扩增３８个循环；最后在７２℃延伸７ｍｉｎ。应用上述反应体系进行板栗 ＲＡＰＤ反应，扩增产物在１．０％琼脂糖凝胶中电泳，经ＥＢ染色后在紫外灯下观察照相，可获得满意的ＤＮＡ 指纹图谱。

Many factors influence stability and accuracy of the result in RAPD reaction. Reaction conditions of RAPD on 8 main cultivated Castenea mollissima clones in Zhejiang province showed the ideal reaction condition: 100 m mol/L This-HCI, 500 m mol/L KCL, 20 m mol/L MgCl2, 0.01% Gelatin, 100 μ mol/L dATP, dTTP, dCTP and dGTP in 20 μ l reaction content with 50-200 ng primer, 0.75-l.0 unit Taq enzyme and 2-10 ng template DNA; reaction condition: 2 minutes Pre-denaturation at 94℃, enlarging 38 circulation at 94℃ in 30 seconds, at 40℃ in 30 seconds and at 72℃ in 1.5 minute; at last, elongation at 72℃ in 7 minutes. RAPD reaction of Castanca mollissima with above-mentioned reaction system, and electrophoresis in l.0% agar-gel of enlarged material, observing and taking photos under ultraviolet lamp after EB staining, satisfied DNA finger print could be obtained.