用复制酶基因cDNA探针检测表达病毒外壳蛋白基因马铃薯中的PLRV

Detection of PLRV in Transgenic Potato Expressing Coat Protein Gene by RdRp cDNA Probe

利用马铃薯卷叶病毒（ＰＬＲＶ）复制酶基因３′端长约６００ｂｐ的ｃＤＮＡ，用缺口平移方法进行３２Ｐ同位素标记，制备成ｃＤＮＡ探针，通过核酸斑点杂交，对提纯的马铃薯卷叶病毒（ＰＬＲＶ）、病毒ＲＮＡ、ＰＬＲＶ感染的马铃薯汁液，表达ＰＬＲＶＣＰ基因的马铃薯叶片及块茎芽进行了检测．结果表明，３２Ｐ标记的复制酶基因ｃＤＮＡ探针特异性强、灵敏度高．可测得提纯病毒的最低含量为４０８ｎｇ／ｍｌ，病毒ＲＮＡ最低量为２７．８ｐｇ／ｍｌ，未转ＣＰ基因接种ＰＬＲＶ的马铃薯叶片提取液的最高稀释度为１３７５．接种ＰＬＲＶ的转ＣＰ基因的抗性马铃薯块茎芽和叶片提取液的最高稀释度为０～１／１５．此探针和只转入病毒外壳蛋白基因，不接种ＰＬＲＶ的转基因马铃薯汁液不发生反应．表明，探针只与ＰＬＲＶ基因组ＲＮＡ特异反应，而不与外壳蛋白基因及其ｍＲＮＡ反应，从而为表达ＣＰ基因的转基因马铃薯中ＰＬＲＶ的检测和抗病性鉴定建立了一种可靠的灵敏的分子生物学技术．此项技术已用于转ＣＰ基因马铃薯Ｄｅｓｉｒｅ，Ｆａｖｏｒｉｔａ。

The 3′end cDNA of putative RNA dependent RNA polymerase gene (RdRp) of Potato leafroll virus (PLRV) was labelled with 32 P and used as cDNA probes for detection of PLRV in transgenic potato plants expressing viral coat protein gene by nucleic acid spot hybridization (NASH).Results show that the purified PLRV can be detected as less as 408 ng/ml,the minimum amount of purified viral RNA detectable was 27.8 pg/ml.The maximum dilution of PLRV infected potato and Non transformed potato leaf extracts were 1∶375.The maximum dilution of PLRV inoculated transgenic potato leaf and tuber sprouts extract was 0-1/15.The 32 P cDNA probes of RdRp does not react with extract from transgenic potato expressing viral coat protein gene and their mRNAs.Tests show that 32 P cDNA probe of PLRV RdRp is sufficient sensitive,specific and useful for detection of PLRV in potato plants,especially in CP gene transformed potato plants.A more sensitive and relatively quantitative molecular biological method for detection of PLRV in transgenic potato expressing viral coat protein gene rather than ELISA was developed and has been used for identification of CP mediated virus resistance.