导　　师： 孙敏;乔爱民

学科专业： G1001

授予学位： 硕士

作　　者： ;

机构地区： 西南师范大学

摘　　要： 随着分子生物学的迅猛发展,rapd(随机扩增多态性dna)标记技术已广泛应用于生物学诸多领域.该文应用rapd技术对黄瓜杂交种子纯度进行了鉴定,并对rapd技术的最适条件进行了探讨.分析了模板dna的制备与不同组织材料对rapd的影响.利用改良sds方法提取黄瓜干种子与一步法提取的黄花苗总dna进行rapd扩增,其结果与传统的ctab和sds的结果一样.这两种方法不仅节约成本,且简单快速大大降低了dna的提取难度,适宜黄瓜杂交种子纯度鉴定.同时证明了不同组织材料对rapd无影响.研究了rapd影响因素.结果表明:mg<'2+>浓度的范围在1.5-3.0mmol.l-1;dntps浓度范围为0.15-0.25mmol.l-1;taq聚合酶浓度范围为0.5-1.5u;模板dna浓度为20-100ng/反应以及引物浓度为0.2-0.4umol.l-1.此外,在对rapd反应体系优化时发现不同厂家以及不同生产时期的taq聚合酶对rapd有较大影响;引物的碱基组成以及不同存放时间的引物对rapd技术也影响. With the rapid development of Molecular Biology, RAPD /(Random Amplified Polymorphic DNA/) has been applied widely in Biological fields. This thesis focused on the application of RAPD to the purity identification of Cucumber hybrid seeds and researched into the optimal conditions of RAPD. It was analyzed that methods to extract DNA template and different tissues had effects on RAPD. The RAPD products with total DNA extracted by improved SDS method from dry seeds and by 'one-step' method from the yellowing young plants were the same as those with total DNA extracted by the traditional CTAB and SDS. Being simple and rapid, two methods could reduce the cost and make extraction of DNA very easily; thus they were suitable for the purity identification of Cucumber hybrid seeds. At the same time, It was shown that different tissues had no influence on RAPD. The paper also discussed the factors that influenced RAPD. The results showed that the best 2~i for RAPD was 1.5-3.0 mmcl. L1 the best dNTPs concentration was 0.1 5-0.2SmmoI. L1 suitable quantity of Taq polymerase 0.5-1 .5U; suitable template DNA quantity 20-1 OOng every reaction and the best concentration of random primer 0.2-0.4 ~i mcI. V'. In addition, it was further found that Taq polymerase with different factories and periods had effects on RAPD when the reaction system of RAPD was optimized, and that there were effects of random primers with different bases and periods on RAPD. The reaction procedure was improved and optimized. The reaction pre-denatured DNA at 940Cfor 4mm, denatured DNA at 940C for 30sec, annealed primers at 370C for 30sec, extended primers at 720C for 1mm, re-extended reaction at 720C for 7mm after 35 cycles and then stored the products at 4 0C. The variety homogeneity was identified and the parental polymorphisms were analyzed. The paper identified the homogeneity of the parent seeds and the hybrid seeds and analyzed the polymorphisms of the parents before screening the primers. The results were that the three false seeds were found among 30 Cucumber hybrid seeds 3 E?1~itZ 1tJ~/(Cucurnis sativus L. ~*PPflNi~J RAPD ~ with three primers S 100, Si 133 and Si 151. The parental homogeneities were very good because the fingerprints of RAPD were the same among 7 female seeds and 7 male seeds. Therefore, there were no false seeds among them. It was shown that the parents had abundant polymorphisms when their polymorphisms were identified with 6 primers S48, 5171, S443, 51019, 51062 and 51357. The primers, which were suitable for the purity identification of Cucumber hybrid seeds, were screened and the fingerprints of RAPD were established among the parents and their hybrid seeds in Cucumber. 32 primers were screened from 102 the ones that could be beneficial to the purity identification of Cucumber hybrid seeds. The results showed that there were 13 primers of partial female type, 9 primers of partial male type, 5 primers of complementary type, 2 primers of peculiar type and 3 primers of shortage type. Hence, their percentages were 12.74/%, 8.82/%, 4.90/%, 1.96/% and 2.94/% respectively. It was found there were 1 female seed, 2 male seeds and 6 false seeds among 94 Cucumber hybrid seeds when the purity of Cucumber hybrid seeds was identified with 4 primers S287, 5293, S297 and S300. Therefore, the purity of the Cucumber hybrid seeds was 90.04/%. The fingerprints of RAPD were established for ZhaoQing No.2 Female SystemT03, ZhaoQing No.2 Male System B35 and their hybrid seeds in Cucumber. As a result, the paper shows that RAPD can be applied to purity identification of the crop seeds and that RAPD has good reliability and accuracy after the reaction system and the reaction procedure are established.

关 键 词： 鉴定 杂种纯度 黄瓜

分 类 号： [S642.2]

领　　域： [农业科学] [农业科学]