广西林业科学
廣西林業科學
엄서임업과학
GUANGXI FORESTRY SCIENCE
2014年
4期
343-350
,共8页
国槐%SRAP-PCR%体系优化%引物筛选%毛细管电泳%正交设计
國槐%SRAP-PCR%體繫優化%引物篩選%毛細管電泳%正交設計
국괴%SRAP-PCR%체계우화%인물사선%모세관전영%정교설계
Sophora japonica%SRAP-PCR%optimization%primer screening%capillary electrophoresis%orthogonal design
为建立国槐(Sophora japonica)最佳SRAP-PCR反应体系,采用正交试验设计L16(45)对影响PCR体系的5个因素(模板浓度、引物浓度、 Mg2+浓度、 Taq聚合酶和dNTP用量)4个水平进行了优化,确定了国槐的最优SRAP-PCR反应体系: Taq聚合酶1.5 U, dNTPs 0.30 mmol/L,模板DNA 90 ng, Mg2+2.0 mmol/L,引物0.3μmol/L, ddH2 O补至20μL。各因素对PCR反应的影响从大到小依次为: Taq聚合酶>dNTPs>模板DNA> Mg2+>引物。基于最佳SRAP体系,采用4个品种对120对引物进行筛选,共获得12对多态性好的引物组合。随机抽取两对引物基于毛细管电泳技术对此体系进行稳定性检验,扩增条带清晰、稳定、多态性高。该体系的建立以及筛选的引物组合为为进一步利用SRAP技术对国槐的遗传多样性、品种鉴定等方面的研究奠定基础。
為建立國槐(Sophora japonica)最佳SRAP-PCR反應體繫,採用正交試驗設計L16(45)對影響PCR體繫的5箇因素(模闆濃度、引物濃度、 Mg2+濃度、 Taq聚閤酶和dNTP用量)4箇水平進行瞭優化,確定瞭國槐的最優SRAP-PCR反應體繫: Taq聚閤酶1.5 U, dNTPs 0.30 mmol/L,模闆DNA 90 ng, Mg2+2.0 mmol/L,引物0.3μmol/L, ddH2 O補至20μL。各因素對PCR反應的影響從大到小依次為: Taq聚閤酶>dNTPs>模闆DNA> Mg2+>引物。基于最佳SRAP體繫,採用4箇品種對120對引物進行篩選,共穫得12對多態性好的引物組閤。隨機抽取兩對引物基于毛細管電泳技術對此體繫進行穩定性檢驗,擴增條帶清晰、穩定、多態性高。該體繫的建立以及篩選的引物組閤為為進一步利用SRAP技術對國槐的遺傳多樣性、品種鑒定等方麵的研究奠定基礎。
위건입국괴(Sophora japonica)최가SRAP-PCR반응체계,채용정교시험설계L16(45)대영향PCR체계적5개인소(모판농도、인물농도、 Mg2+농도、 Taq취합매화dNTP용량)4개수평진행료우화,학정료국괴적최우SRAP-PCR반응체계: Taq취합매1.5 U, dNTPs 0.30 mmol/L,모판DNA 90 ng, Mg2+2.0 mmol/L,인물0.3μmol/L, ddH2 O보지20μL。각인소대PCR반응적영향종대도소의차위: Taq취합매>dNTPs>모판DNA> Mg2+>인물。기우최가SRAP체계,채용4개품충대120대인물진행사선,공획득12대다태성호적인물조합。수궤추취량대인물기우모세관전영기술대차체계진행은정성검험,확증조대청석、은정、다태성고。해체계적건립이급사선적인물조합위위진일보이용SRAP기술대국괴적유전다양성、품충감정등방면적연구전정기출。
Orthogonal experimental design of L16 (45 ) was applied to establish the optimal reaction sys-tem for SRAP-PCR in terms of five factors ( template, primer, Mg2+, Tap polymerasease and dNTPS) at four levels. The optimal SRAP-PCR system was obtained as follows: Tap polymerasease 1. 5 U, dNTPs 0. 30 mmol/L, template DNA 90 ng, Mg2+ 2. 0 mmol/L, each primer 0. 3μmol/L, ddH2 O filled to 20μL. The results indicated that the five factors affecting PCR reaction in a descending order as:Tap poly-merasease, dNTPs, template DNA, Mg2+, primers. Out of the 120 pairs of SRAP primer combinations screened based on this optical system, 12 pairs produced a high level of polymorphism. Two pairs of primers were random slected for stability test based on capillary electrophoresis technology, and these am-plified bands were clear, stable and high polymorphism. These findings showed that the optimized SRAP-PCR system and primer combinations screened could be used for variety identification and genetic diversi-ty analysis of Sophora japonica .