三种单链特异性核酸酶检测桉树ESTP的酶切条件优化  

Optimization of Digestion Conditions with three Single-strand Specific Nucleases for Detection of Expressed Sequence Tag Polymorphisms (ESTP) in Eucalyptus

作　　者：郭勇[1] 李发根[1] 王宇[1] 张晓红[1] 李梅[1] 甘四明[1] 

机构地区：[1]中国林业科学研究院热带林业研究所,广州510520

出　　处：《分子植物育种》2008年第3期608-614,共7页Molecular Plant Breeding

基　　金：国家自然科学基金(30671703);863计划(2006AA100109)

摘　　要：单链特异性(SSS)核酸酶是基因组学研究的有效工具。本研究对绿豆芽核酸酶、S1和芹菜汁提取物三种SSS核酸酶检测桉树ESTP检测的酶切条件进行了优化,优化因子依次包括Mg2+浓度、酶切温度、酶用量和/或pH值等因子。三种酶的最适酶切温度均为60℃。绿豆芽核酸酶优化后的酶切反应体系为:10×Buffer(pH5.56)1.0μL(NaAc300mmol/L,Nacl1.0mol/L,ZnAc210mmol/L,甘油50%,MgCl2100mmol/L),PCR产物5μL,酶4.5U,超纯水补至10μL;S1的优化酶切体系为:10×Buffer(pH5.46-5.67)1.0μL(MgCl2100mmol/L,ZnAc22mmol/L,Bis-Tris200mmol/L,TritonX1000.02%,BSA0.002mg/mL),PCR产物5μL,S1酶5U,超纯水补至10μL;CJE的优化酶切体系为:10×Buffer(pH7.5)1.0μL(MgCl2100mmol/L,Hepes100mmol/L,KCl100mmol/L,TritonX1000.02%,BSA0.002mg/mL),PCR产物5.0μL,CJE酶1.0μL,超纯水3.0μL。综合考虑酶切效果和成本,推荐S1为检测桉树ESTP的经济、有效的SSS核酸酶。Single-strand specific （SSS） nucleases provide important tools for genomics studies. We optimized in this paper the digestion conditions with mung bean nuclease, S 1 and CJE for detection of expressed sequence tag polymorphisms （ESTP） in Eucdyptus, including Mg^2＋ concentration, temperature, nuclease dosage, and/or pH value. The optimal temperature was exclusively 60℃ for all the three nucleases. Mung bean nuclease could result in optimal digestion with a 10 μL reaction including 10xBuffer （pH 5.56） 1.0 μL （NaAc 300 mmol/L, NaCI 1.0 mol/L, ZnAc2 10 mmol/L, glycerol 50%, and MgCI2 100 mmol/L）, PCR product 5 μL, the nuclease 4.5 U, and water added to a total volume of 10 μL. S 1 followed the below optimal reaction, 10xBuffer （pH 5.46-5.67） 1.0μL （MgCI2 100 mmol/L, ZnAc2 2 retool/L, Bis-Tris 200 mmol/L, Triton X100 0.02%, and BSA 0.002 mg/mL）, PCR product 5 μL, S1 5 U, and water supplemented to 10 μL. The digestion reaction for CJE optimized is 10 Buffer （pH 7.5） 1.0μL （MgCI2 100 mmol/L, Hepes 100 mmol/L, KCI 100 mmol/L, Triton X100 0.02%, and BSA 0.002 mg/mL）, PCR product 5.0 μL, CJE 1.0 μL, and water 3.0 μL. It is recommended that S1 is the most economical and applicable choice ofSSS nuclease for eucalypt ESTP detection.

关 键 词：单链特异性核酸酶 条件优化 ESTP 桉树 

分 类 号：S792.39[农业科学—林木遗传育种]