应用Digoxigenin标记的cDNA探针检测香蕉束顶病毒  

Detection of Banana Bunchy Top Virus by Nucleic Acid Spot Hybridization with Digoxigenin Labeled cDNA Probe

作　　者：孟清[1,2] 曹先维[1,2] 张彤[1,2] 

机构地区：[1]内蒙古大学生物学系 [2]华南农业大学园艺系

出　　处：《内蒙古大学学报（自然科学版）》1999年第3期367-371,共5页Journal of Inner Mongolia University：Natural Science Edition

基　　金：广东省自然科学基金

摘　　要：用非放射性的Ｄｉｇｏｘｉｇｅｎｉｎ标记的１１－ｄＵＴＰ取代３２Ｐ标记的ｄＡＴＰ或ｄＣＴＰ，标记克隆的ＢＢＴＶｃＤＮＡ－１０．５ｋｂｃＤＮＡ，制备成探针．通过核酸斑点杂交对粗提取的ＢＢＴＶ，ＢＢＴＶ－ＤＮＡ，感染ＢＢＴＶ的香蕉植物的拟茎，球茎处的组织，新生叶片以及成熟后的叶片进行了检测．结果表明：ＢＢＴＶｃＤＮＡ－１ｃＤＮＡ探针特异性强，不与ＣＭＶ—ＲＮＡ、无毒香蕉组培苗提取的核酸发生杂交反应；仅与粗提ＢＢＴＶ、ＢＢＴＶ－ＤＮＡ、ＢＢＴＶ侵染的香蕉组织的核酸提取物发生杂交反应．此探针灵敏度高，可检出含有ＢＢＴＶｃＤＮＡ－１０．５ｋｂ的质粒ＤＮＡ的最小量为１０ｐｇ；测定感病香蕉植株的拟茎汁液的最高稀释度可达１∶１２８，相当于０．４ｍｇ病蕉组织中的病毒含量．测定同一病株不同部位的结果表明，ＢＢＴＶ在植物体内的分布不均匀，拟茎处组织中病毒含量最高，球茎处的组织以及新生叶片中的病毒含量次之。Plasmid pBTR 12 which contains 0.5 kb sequence of BBTVc DNA 1 was used as a probe labeled with digoxigenin 11 dUTP.The plasmids pBTR 12 ,pBTR100,crude purified BBTV particles,BBTV DNA,the extract of BBTV infected banana Pseudostem,Corm/meristem,new leaves,old leaves were detected by the nucleic acid spot hybridization,the detection is accomplished follow by enzymatic reaction of alkaline phosphatase,conjugated to anti digoxigenin antibodies.The tests show that the BBTV DNA probe only hibridized to samples from BBTV infected banana tissue,BBTV,BBTV DNA and not from CMV RNA and healthy banana tissue.The maximum dilution of BBTV infected banana Pseudostem extracts which gives positive reaction is 1∶128,which is equivalent to 0.4 mg banana tissue.The tests also indicated that the distribution of BBTV within plants was not homogeneous.The concentration of virus appeared to higher in Pseudostem region,lower in corm/meristem regions and new leaves,much lower in older leaves.

关 键 词：香蕉束顶病毒 CDNA探针 非放射性检测 病毒 

分 类 号：S436.68[农业科学—农业昆虫与害虫防治] S432.41[农业科学—植物保护]