【作者】 姚湧；

【导师】 夏立照； 汪学龙；

【作者基本信息】 安徽医科大学 ， 病原生物学， 2001， 硕士

【摘要】 目的 应用十二烷基硫酸钠—聚丙烯酰胺凝胶电泳（SDS—PAGE）和随机扩增多态性DNA（RAPD）技术获取安徽省三种常见蜚蠊的可溶性蛋白质区带和DNA指纹图谱，对三种常见蜚蠊的可溶性蛋白质和基因组DNA多态性进行分析研究，为蜚蠊分类提供科学依据。方法 采集安徽省三种常见蜚蠊黑胸大蠊、美洲大蠊、德国小蠊成虫标本，进行形态学的分析鉴定。首先应用冻融匀浆法制备这三种蜚蠊的可溶性蛋白质匀浆液，进行十二烷基硫酸钠—聚丙烯酰胺凝胶电泳（SDS—PAGE），获取三种常见蜚蠊的可溶性蛋白质区带，用考马斯亮蓝G-250染色后，观察和计算出各蜚蠊主要蛋白质区带的分子量。然后，提取三种蜚蠊的基因组DNA，进行鉴定及定量分析。确定随机扩增多态性DNA（RAPD）反应总体积、反应条件。选取5条不同的随机排列碱基顺序的多聚核苷酸单链为引物，进行RAPD-PCR扩增反应。将扩增产物进行琼脂糖凝胶电泳，获得这三种蜚蠊的DNA图谱，计算出三种蜚蠊种间的遗传距离。最后对这三种蜚蠊可溶性蛋白质区带和基因组DNA多态性分析和研究。结果 用十二烷基硫酸钠—聚丙烯酰胺凝胶电泳（SDS—PAGE）进行研究显示：黑胸大蠊（P.f.）雄虫和雌虫蛋白条带数目分别是29条、31条；美洲大蠊（P.am.）雄虫和雌虫蛋白条带数目分别是28条、30条；德国小蠊（B.g.）雄虫和雌虫蛋白条带数目分别是25条、28条。通过比较发现三种蜚蠊的蛋白质区带数目和位置有差别；B.g主要蛋白质区带的位置与两种大蠊相差明显，雌虫与雄虫之间也有差别。用随机扩增多态性DNA（RAPD）进行研究显示：这三种蜚蠊分别被扩增出不同数量和分子大小的DNA片段。三种蜚蠊基因 安徽医科大学硕士研究生学位论文 组DNA扩增产物中具有共同的扩增条带，这反映了三种蜚蟋基因组DNA具有同 源性；同时三种蜚烧基因组DNA扩增产物又具有各自独特的DNA条带，DNA 条带亮度上也有差别。用引物 OPA｛扩增，美洲大镰m．）在 0．73kb、0．40kb 处条带较亮；黑胸大镰（＊f）在0．70kb处条带较亮：德国小烧（B．g．）在0．52kb 处条带较亮。用引物＊PA－ic扩增，美洲大镰（P二．）在队33讪处较亮：黑胸大 螨（Rf）在 0．80kb处较亮；德国小镰（B．g．）在 0．33kb处条带较亮。用引物 OPA－15 扩增，美洲大镰（Ram．）在0．50kb、0．70kb处较亮；黑胸大镰（Pf）在0了okb和 0．85kb处较亮；德国小镰（B．g．）在0．32kb、0．53kb、0．6Ikb、0．85kb、l＊okb、1．10kb 和1．20kb处条带较亮。用引物o PAPA－16扩增，美洲大蛾（R＿．）在0．30kb、0．40kb、 O．50kb处条带较亮：黑胸大镰（尺f）在0．40kb、O．70kb处条带较亮；德国小镰（B．g．） 在0．40kb、0．94kb处条带较亮。用引物OPA－19扩增，美洲大琐（Ram．）在0．38kb、 O．52kb处条带较亮；黑胸大镰（Pf）在0．45kb0．52kb处条带较亮；德国小烧旧召．） 在0．52kb、0．85kb、l．00kb、l．20kb处条带较亮。美洲大镰与黑胸大峻遗传距离为 0．3257较近：德国小螨与黑胸大镰和美洲大螨的遗传距离分别为0石276和0．6729 较远。结论三种常见蜚镰的可溶性蛋白质区带和DNA指纹图谱大部分相同但 存在差异，这种差异可为蜚镰鉴定提供分类依据。SDS—PAGE和RAPD用于蜚焕 种类的鉴定，显示了较好的特异性和准确性，并因其简便和快速，可为蜚贱提供 新的分类方法。更多还原

【Abstract】 Objective To find scientific classification methods for cockroach analysis of discrepancies among three ordinary species of cockroaches in Anhui province has been made by comparing the soluble protein strips and the genomic DNA polymorphic . The soluble protein strips and the genomic DNA fingerprints of three ordinary species of cockroaches in Anhui province were gained by using SDS-PAGE and RAPD technique. Methods The specimens of cockroaches were collected in Anhui province , and were identified by morphology . Firstly the soluble protein liquid were made by the way of freeze and melt Homogenate. SDS-PAGE was carried out in the liquid. Dyed by Coomassie brilliant blue G250 , the molecular weight of main protein strips of the cockroaches were measured and calculated by standard curve . Secondly the Genomic DNA of the three ordinary species of cockroaches was extracted, and identified and quantitative analysed .The total volume of reaction and conditions of RAPD was defined . Five primers with different arbitrary single-chain polynucleotide sequences were selected. The groups of DNA were amplified by RAPD , and the DNA fingerprint maps were gained by running agarose gel electrophoresis . the genetic distances among three ordinary species of 6 cockroaches were calculated . Lately the soluble protein strips and the genomic DNA polymorphic of three ordinary species of Cockroaches were studied and analysed Results The SDS-PAGE experiment suggested there exist some discrepancies More than 20 strips were identical on the SDS-PAGE gel the numbers of female and male of P.f. are 31 strips and 29strips ; the numbers of female and male of P.am. are 30strips and 28strips; the numbers of female and male of B.g. are 28strips and 25strips . the numbers and locations of protein bands were different among these species . It was also obvious that the positions of the main protein bands in B.g. were different from those in two Periplaneta species , and the positions of the main protein bands in female insect were different from those in male insect Little difference was founded in the bands of two Perz~planeta species with only a slightly different appearance in their densities . The RAPD experiment suggested there exist some discrepancies in the amplified DNA fragments of three species. The DNA maps that the numbers and size of DNA bands were different among the species have gained by RAPD . The common fragments indicated some degree homogeneity of three species genomes , their distinctive fragments and intensity suggested the diversity among the species . The sizes of bands in P.am. that were more intensive than others obtained by primer OPA-8 were 0.73kb and 0.40kb , the bands in P.f. were 0.70kb , the bands in B.g. were 0.52kb. The sizes of bands in P.am. that were more intensive than others obtained by primer OPA-1O were 0.33kb , the bands in P.f. were 0.80kb , the bands in B.g. were 0.33kb . The sizes of bands in P.am. that were more intensive than others obtained by primer OPA-15 were 0.50kb and 0.70kb , the bands in P.f. were 0.70kb and 0.85kb , the bands in B.g. were 0.32kb. 0.53kb. 0.61kb. 0.85kb. 1.00kb. 1.10kb and 1.20kb . The sizes of bands in P.am. that were more intensive than others obtained by primer OPA-16 were 0.30kb.更多还原