百合三种主要病毒的RT-PCR检测及脱毒技术研究  

Study on Molecular Detection and Elimination of Three Lily(Lilium longiflorum) Viruses

作　　者：陈进[1] 李晓昕[1] 袁雪[1] 易瑾[1] 罗弦[1] 赵阳[1] 义鸣放[1] 

机构地区：[1]中国农业大学观赏园艺与园林系,北京100193

出　　处：《农业生物技术学报》2013年第4期489-497,共9页Journal of Agricultural Biotechnology

基　　金：农业部公益性行业(农业)科研专项(No.200903020);农业部"948"引进项目(No.2011-G17)

摘　　要：百合病毒病是影响百合切花和种球产量与质量的关键问题之一,虽然在我国百合病毒检测和脱毒技术的研究已经展开,但是在病毒检测中存在着检测病毒的种类较单一和检测结果出现假阴性等问题;脱毒方面也存在操作难度大、脱毒率不高等缺陷。本研究以麝香百合杂种系品种雷山一号(Lilium longiflorum cv.Raizan 1)组培苗为试材,对侵染百合的3种主要病毒:黄瓜花叶病毒(Cucumber mosaic virus,CMV)、百合无症病毒(Lily symptomless virus,LSV)和百合斑驳病毒(Lily mottle virus,LMoV)的多重RT-PCR检测及病毒脱除技术进行了研究。根据百合18SrRNA和3种病毒的基因序列,设计了4对特异引物,并对多重RT-PCR中的dNTPs浓度、Mg2+浓度、TaqDNA聚合酶浓度、退火温度和反应循环数等因素进行了筛选和优化。结果表明,进行多重RT-PCR的最佳反应体系为:dNTPs(2.5mmol/L)1.5μL,Mg2+(25mmol/L)1.5μL,Buffer1.875μL,cDNA1μL,CMV、LMoV、LSV、18S上、下游引物各0.5μL,TaqDNA聚合酶0.2μL,加H2O补足至25μL。最佳反应程序为:94℃预变性5min;94℃变性30s,52.4℃退火30s,72℃延伸30s,30个循环;72℃延伸10min,4℃终止反应。由此建立了以百合18SrRNA为内参照,同时检测CMV、LSV和LMoV的多重RT-PCR技术。黑暗、变温热处理结合试管鳞茎培养只对脱除百合植株CMV有效,脱毒率达90%以上;黑暗、变温热处理后,切取≤1mm的茎尖进行光培养能够有效脱除CMV、LSV和LMoV3种病毒,脱毒率达到93.3%。此脱毒方法具有操作简便、培养周期较短、脱毒率高等优点,可以为百合脱毒苗的生产提供指导。Viral diseases cause adverse effects on the yield and quality of lilies grown in China. However, we still can＇t detect and eliminate these viruses effectively. The viral detection methods used these days are either time-consuming and expensive or sometime provide false-negative results occasionally. On the other hand, difficulties in operation mechanisms and low virus-free rate are the major constraints restricting the development of virus elimination techniques. Addressing these limitations, we carried out this study to establish effective ways to detect and eliminate three major viruses in lilies namely, Cucumber mosaic virus （CMV）, Lily symptomless virus（LSV） and Lily mottle virus（LMoV）. In vitro plantlets of Lilium longiflorum cv. Raizan 1 were used as subject material. Specific primer pairs were designed according to the gene sequence of the three viruses and 18S rRNA, and used for multiplex RT-PCR. Several reaction components （dNTPs, Mg^（2＋） and Taq DNA polymerase）, annealing temperature and cycle numbers were optimized to conduct multiplex RT- PCR effectively. Among couple of used reaction systems, the most effective and precise reaction system was as follow： dNTPs（2.5 mmol/L） 1.5 μL, Mg^（2＋）（25 mmol/L） 1.5 μL, Buffer 1.875 μL, eDNA 1 μL, Taq DNA polymerase 0.2 μL and each primer 0.5 μL, adding H20 to a final volume of 25 μL. The optimal reaction procedure wasas follow： 94% denaturation temperature for 5 min;94℃ for 30 s,52.4℃annealing temperature for 30 s,extension phase with 72℃ for 30 s,30 PCR cycles;72℃ for 10 min,and kept at 4℃ to terminate the reaction. Thus, we established an efficient method to detect the three viruses simultaneously. The 18S rRNA was used as an internal control to avoid false-negative during the procedure. Heat treatment （38℃ for 14 h, 32℃ for 10 h, all in dark） combining with bulblet culture could eliminate more than 90% of the CMV in lilies, but remains ineffective in eliminating LMoV or LSV. After heat treatment

关 键 词：百合 多重RT-PCR 病毒检测 脱毒 

分 类 号：S852.659.5[农业科学—基础兽医学]