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[摘要]
目的探讨影响青葙SRAP(sequence-related amplified polymorphism)扩增的各种因素,建立并优化青葙SRAP反应体系,为分子水平鉴定青葙子及其伪品,探讨青葙不同种质间的亲缘关系奠定基础。方法用CTAB法提取青葙DAN,设计Taq酶浓度(0.02、0.04、0.06U/μL)、dNTP浓度(0.10、0.20、0.30mmol/L)、Primer浓度(0.15、0.30、0.45μmol/L)3水平3因素试验和Mg2+浓度(0.5、1.0、1.5、2.0、2.5、3.0、3.5、4.0mmol/L)8水平单因素试验。在25μL体系中加入模板DNA20ng,对体系进行优化,用琼脂糖进行检测。结果建立了适合青葙的SRAP体系,在25μL体系中,DNA为20ng,Taq酶浓度为0.04U/μL,dNTP浓度为0.30mmol/L,Primer浓度为0.30μmol/L,Mg2+浓度为2.5mmol/L,优化后的体系目标条带增多,且比较稳定,重现性好,得到了较好的扩增效果。结论本研究建立的反应体系适合青葙SRAP的研究。为从分子水平鉴定青葙子正品与伪品以及鉴定青葙不同种质资源奠定了基础。
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[Abstract]
Objective To study the factors of influencing the amplification of sequence-related amplified polymorphism(SRAP) system in Celosia argentea,and to develop and optimize the SRAP reaction system,laying a foundation for identifying spurious breed at molecular level and researching genetic relationships between different germplasms of C.argentea.Methods Cetyl-trimethyl-ammonium-bromide(CTAB) method was used to extract the genomic DNA of C.argentea.The tests with three factors at three levels were designed,including Taq polymerase concentrations(0.02,0.04,and 0.06 U/μL),dNTP concentrations(0.10,0.20,and 0.30 mmol/L),and Primer concentrations(0.15,0.30,and 0.45 μmol/L).Furthermore,eight tests were designed based on the single factor of Mg2+ concentrations(0.5,1.0,1.5,2.0,2.5,3.0,3.5,and 4.0 mmol/L).DNA Template(20 ng) was added into 25 μL SRAP reaction system,which was optimized.Agarose electrophoresis was used for detection.Results An efficient SRAP reaction system for C.argentea was developed.The optimum system was as followes:DNA(20 ng) Taq polymerase 0.04 U/μL,dNTP 0.30 mmol/L,Primer 0.30 μmol/L,and Mg2+ 2.5 mmol/L.The total volume for reaction was 25 μL.Target bands were stable with good reproducibility after optimization and the amplification results were satisfactory.Conclusion The SRAP reaction system in this study is suitable for analysis of C.argentea,laying a foundation for identification of false from genuineness at the molecular level and genetic research among different germplasms of C.argentea.
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