INFECTIOUS CUCUMBER MOSAIC-VIRUS RNA TRANSCRIBED INVITRO FROM CLONES OBTAINED FROM CDNA AMPLIFIED USING THE POLYMERASE CHAIN-REACTION
INFECTIOUS CUCUMBER MOSAIC-VIRUS RNA TRANSCRIBED INVITRO FROM CLONES OBTAINED FROM CDNA AMPLIFIED USING THE POLYMERASE CHAIN-REACTION
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DOI：
10.1099/0022-1317-71-11-2503
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发表时间：
1990-11-01
影响因子：
3.8
通讯作者：
BUCK, KW
中科院分区：
文献类型：
--
作者：
HAYES, RJ;BUCK, KW
采用聚合酶链反应（PCR）扩增了黄瓜花叶病毒Q株（CMV-Q）RNA 1、RNA 2和RNA 3的全长cDNA。第一链引物含有BamHI位点和与RNA的3“末端互补的序列。第二链引物含有BamHI位点、T7启动子和对应于每个RNA的5“末端的序列。用BamHI酶切后，将PCR产物克隆到载体pEMBL 9（+）的BamHI位点。选择每种RNA的五个克隆，并使用T7 RNA聚合酶从每个克隆体外合成RNA转录物。构建体被设计为允许转录精确地在每个RNA的5“末端起始。所有的转录本接种到烟草（Nicotiana tabacum cv.）Samsun植物三个一组，分别对应于RNA 1、RNA 2和RNA 3。在转录物组中，四种诱导的症状与CMV-Q RNA诱导的症状不可区分。然而，第五个转录物组诱导了严重得多的症状。还构建质粒以允许合成在每个RNA的5“末端具有一个或两个额外G残基的转录物。尽管用T7 RNA聚合酶体外合成的此类转录物的产率更高，但它们的感染性低于5 ''末端没有额外残基的转录物。
Full-length cDNA to RNA 1, RNA 2 and RNA 3 of cucumber mosaic virus strain Q (CMV-Q) was amplified using the polymerase chain reaction (PCR). The first-strand primer contained BamHI site and sequences complementary to the 3'' terminus of the RNA. The second-strand primers contained a BamHI site, a T7 promoter and sequences corresponding to the 5'' terminus of each RNA. After cleavage with BamHI, the PCR products were cloned into the BamHI site of the vector pEMBL9(+). Five clones of each RNA were selected and RNA transcripts were synthesized in vitro from each clone using T7 RNA polymerase. The constructs were designed to allow transcription to initiate precisely at the 5'' terminus of each RNA. All the transcripts were found to be infectious when inoculated onto Nicotiana tabacum cv. Samsun plants in sets of three, corresponding to RNA 1, RNA 2 and RNA 3. Of the transcript sets, four induced symptoms indistinguishable from symptoms induced by CMV-Q RNAs. However a fifth transcript set induced much more severe symptoms. Plasmids were also constructed to allow synthesis of transcripts with one or two additional G residues at the 5'' terminus of each RNA. Although the yields of such transcripts synthesized in vitro with T7 RNA polymerase were higher, their infectivity was lower than of those with no additional residues at their 5'' termini.