利用正交设计优选菜心ISSR-PCR反应体系.pdfVIP

仲恺农业技术学院学报，21(1)：01～05，2008 Journa／ofZhongkai University ofAgriculture and Technology 文章编号：1006—0774(2008)01—0001—05 Orthogonal design for optimal ISSR—PCR system in flowering Chinese cabbage SUN Xue．mei ， ，QIAO Ai．rain ，SUN Min ，GUI Teng qin ， (1．School of Life Sciences，Southwest University，Chongqing 4007 1 5，China；2．Collage of Agriculture and Landscape，Zhongkai University of Agriculture and Technology，Guangzhou 510225，China) Abstract：Factors which affected the ISSR—PCR reaction of flowering Chinese cabbage(Brassica campes— tris L．ssp．Chinensis Var．utilis Tssen．et Lee．)，such as concentrations of primer，Taq DNA polymer— ase，dNTPs and Mg“ ， was studied by L1 6(4 )orthogonal test．The optimal ISSR．PCR system for flow— ering Chinese cabbage was determined．In a total volume of 25 l，containing 2．5 L 10×buffer，2．0 mmol／L Mg“ 0．5 units of Taq DNA polymerase，0．24 mmol／L dNTPs，0．50／,Lmol／L primer and 6O ， ng of template DNA，clear and reproducibe DNA fragments can be obtained． Key words：Brassica campestris L．ssp．Chinensis Var utilis Tssen．et Lee．；ISSR；orthogonal design CLC number：S634 Document code：A 利用正交设计优选菜心ISSR．PCR反应体系 孙雪梅 ，乔爱民 ，孙 敏 ，桂腾琴 ， (1．西南大学生命科学学院，重庆400715；2．仲恺农业技术学院农业与园林学院，广东广州510225) 摘要：利用正交设计L16(4 )探讨引物、 聚合酶、dNTPs、Mg2 对菜心ISSR—PCR反应的影响．得到适合菜心IS— SR·PCR反应的最佳体系：在25 反应体系中含2．5 10×buffer，2．0 mmol／L M ，0．5U Taq DNA聚合酶， 0．24 mmol／L dNTPs，0．5~rnol／L引物，60 ng DNA模板． 关键词：菜心；ISSR；正交设计 Inter—Simple Sequence Repeat(ISSR)is a technique in which repeat anchored primers are used to amplify DNA sequences between two inverted SSR ．ISSR markers do not require a prior knowledge of the SSR target se— quence，furthermore，they are highly reproducible due to their primer length a