Ying Xu, Chenggang Zhu, Yongfeng Jin, Yaozhou Zhang
1Biochemistry and Molecular Biology Institute of Zhejiang University, 310029 Hangzhou, China.
2Zhejiang University of Science, 310018 Hangzhou, China.
Chin Sci Bull. 2004;49(12):1261-1266. doi: 10.1360/03wc0550. Epub 2013 Aug 30.
RNA interference (RNAi) causes degradation of targeted endogenous RNA in many diverse organisms. To investigate the effect of dsRNA on silkworm cells, we transfected three kinds of synthetic dsRNAs of 435 bp(Ap), 300 bp(Ap) and 399 bp(A) in length against the various regions of BmNPV's DNA polymerase gene and DNA helicase gene, which are indispensable for viral replication in silkworm cells by TransMessenger transfection Reagent. Results indicated that in the experiment where silkworm cells were infected with wild-strain BmNPV of the three dsRNAs, Ap and A can effectively suppress the replication of virus, but Ap had no effect on the inhibition of viral replication. Ap and A can reduce the infective titer of BmNPV with a peak change of approximately 3-4 logs on day 4 post-infection. The results of reverse transcript polymerase chain reaction (RT-PCR) and DNA dot blotting also indicated that the expression level of the two target genes and the quantity of viral DNA both distinctly decreased under the influence of Ap or A. Furthermore, using fluorescence microscopy we analyzed the distribution patterns of dsRNA. Our studies revealed that a majority of dsRNA was localized in the nuclear periphery discontinuously after 24 h of transfection.
RNA干扰（RNAi）在许多不同生物中会导致靶向内源RNA的降解。为了研究双链RNA（dsRNA）对家蚕细胞的影响，我们通过TransMessenger转染试剂转染了三种长度分别为435 bp（Ap）、300 bp（Ap）和399 bp（A）的合成dsRNA，它们分别针对家蚕核型多角体病毒（BmNPV）DNA聚合酶基因和DNA解旋酶基因的不同区域，而这两个基因对于病毒在家蚕细胞中的复制是不可或缺的。结果表明，在三种dsRNA转染家蚕细胞并感染野生型BmNPV的实验中，Ap和A能够有效抑制病毒复制，但Ap对病毒复制的抑制没有效果。Ap和A能够降低BmNPV的感染滴度，在感染后第4天峰值变化约为3 - 4个对数级。逆转录聚合酶链反应（RT-PCR）和DNA斑点杂交结果也表明，在Ap或A的影响下，两个靶基因的表达水平以及病毒DNA的量均明显下降。此外，我们使用荧光显微镜分析了dsRNA的分布模式。我们的研究表明，转染24小时后，大多数dsRNA间断地定位在核周边。
