库尔勒香梨的苹果茎痘病毒cDNA探针检测技术研究  

Studies on cDNA Probe Detection Technology of Apple Stem Pitting Virus in Korla Pear

作　　者：牛建新[1] 朱军[1] 马兵钢[1] 覃伟铭[1] 王小兵[1] 吴忠华[1] 

机构地区：[1]石河子大学农学院园艺系

出　　处：《中国农业科学》2004年第1期99-105,共7页Scientia Agricultura Sinica

基　　金：国家自然科学基金资助项目 ( 3 0 0 60 0 5 3 );教育部科学技术研究重点资助项目 ( 0 2 180 );兵团科委资助项目 (NKB0 2SDXNK0 1SW );国家科技攻关计划引导资助项目 ( 2 0 0 3BA5 46C)

摘　　要：以库尔勒香梨叶片和皮层为材料 ,对 2种总RNA提取方法进行了比较研究 ,从中行筛选出了适合库尔勒香梨的提取方法 ;并利用RT PCR反应体系成功合成了生物素标记的cDNA探针 ,在此基础上 ,对影响杂交灵敏度的主要因素进行了试验研究 ,结果表明 ,当探针浓度为 4 0 0ng·ml-1,甲酰胺浓度为 4 5 % ,4 2℃杂交反应 6h ,可获得最高灵敏度。进口硝酸纤维素膜的灵敏度最好 ,基本无背景。Tween2 0的封闭效果比牛血清白蛋白好。对不同组织和不同提取方法提取的总RNA进行了杂交检测 ,结果表明 ,不论幼叶、老叶、皮层及冷冻叶片均可出现杂交信号 。Using Korla pear leaves and cortexes as materials, total RNA was extracted using two methods, and these methods was compared. The fittest methods for Korla pear were screened out and the also Biotin labeled cDNA Probe was yielded with RT-PCR. Main factors affecting sensitivity of hybridization were studied. Studies indicated that the highest sensitivity was obtained at probe concentration 400 ng·ml -1, 45%formamide and 42℃ for 6 h. The best result of hybridization was obtained by using imported nitrocellulose membrane. Blocking result of Tween20 is better than that of Albumin Bovine. The results of detection of the total RNA of different tissues and extracting methods were compared. Studies indicated that detection of the total RNA of fresh leaf, old leaf, cortexes and freezing leaf and two extracting methods showed signs of hybridization.

关 键 词：库尔勒香梨 苹果茎痘病毒 CDNA探针 检测技术 

分 类 号：S436.611[农业科学—农业昆虫与害虫防治]