【作者】 刘宏；

【导师】 牛建新；

【作者基本信息】 石河子大学 ， 果树学， 2006， 硕士

【摘要】 原位逆转录PCR是一种特异、敏感的分子生物学---细胞学方法,结合了PCR和原位杂交的优点,既可检测出样品中低拷贝的核酸,又可确定其细胞来源和在细胞中的位置。开展该研究,可通过观察扩增的cDNA在组织中的分布,进行病毒感染分析。这样既可为果树病毒检测提供一种新的途径,又可为研究病毒在组织中的生成、分布和转移途径提供帮助,也为果树无毒化生产提供一理论依据。因而具有重要的理论价值和生产实践意义。本试验以已知带有苹果褪绿叶斑病毒的香梨和杜梨实生苗的茎尖为材料,用Dig-11-duTP对扩增产物进行标记。研究了香梨茎尖石蜡切片IS-RT-PCR及原位杂交检测技术。本研究的主要内容包括:1.系统研究了石蜡切片方法对原位RT-PCR及原位杂交切片质量的影响。结果表明:组织经4%多聚甲醛固定1h;50%、70%、85%、95%、100%乙醇脱水各1h;1/2二甲苯和1/2乙醇处理1h一次、二甲苯处理1h一次;浸蜡低温38℃过夜处理;高温58℃2h一次共3次;切片厚度为7-8μm;并用酒精-水浴法展片。适合于以茎尖为材料的原位RT-PCR及原位杂交。2.用PCR法制作出了适合原位杂交的地高辛标记的cDNA探针。3.应用了一步RT-PCR反应,简化了试验步骤,缩短了试验周期。4.确定了梨树苹果褪绿叶斑病毒在茎尖中的分布位置。试验中设置了多种阴性对照,结果表现为:阴性对照组织中均未显现蓝紫色。而阳性材料组织的一些细胞表现出明显的蓝紫色,说明梨树苹果褪绿叶斑病毒在茎尖中有可能主要分布于幼叶的外围皮层组织及相应的初生维管束。此外进行了碱性磷酸酶显色系统可靠性研究,当切片仅在扩增时混合液中不加入地高辛标记的核苷酸而其他处理相同,载片上的组织未显示蓝紫色,这说明结果不会受到内源碱性磷酸的影响,具有较强的特异性。更多还原

【Abstract】 In situ reverse transcipte polymerase chain reaction(In situ RT-PCR) is a rapid and sensitive method for molecule biology and cytology,which was a combination of PCR and in situ hybridization,could examin low copy nucleic acids in tissue,yet could still identify the celluar origin and location of signals。Doing the study has great theoretical and pratical significances,as it would not only offer a kind of new channel for the virus detection of fruit tree,but also would give help in studies of virus’generation,stribution and virus transfer channel。In the experiment, using Kuala pear stem with pear ring pattern mosaic virus and as material, using digoxigenin -duTP as labele to cDNA。And studies on detection of viral disease in kuala pear using in situ PCR and situ hybridization.The paper includes major contents as follow,1.Studied systemically effect of making section manner on quality of in situ PCR.The result shown:it is suitable for using leaves as materials to do In situ PCR that the tissue is fixed in 4% PFA over 1h,dehydrated by 50%、70%、85%、95%、100% ethanol 1h respectively,transparented 1h by xylene,immersed paraffine twice each 2h,embed tissue in parafin cut sections at 7-8μm,and strech sections on ethanol-water.2.Using ACLSV’s special primer paies,Dig labelled cDNA probes are synthesized.3.One-step ISRT-PCR assay is carried out by using Thermus thermophilus DNA polymerase.4. Have determined ACLSV some distribution in stem。In experiment many negative controls were made。The result shows:there are not blue-purple signals in negative control tissue,however,some cell expressions of the mesophyll of positive material tissues make obvious blue purple colour ,which explain ACLSV may distribute in peripheral vascular and cerebral cortex。Moreover,alkaline phosphatase system’s reliablity is studied。The tissues on slide don’t display blue-purple if the mix uninclude digoxigennin labled nucleotide only when the section is amplified,but be done other same treat as the positive。This demonstrate the result can’t be affected by endogenous alkaline phosphatase,and the assay has strong speciality。更多还原