Production of double-stranded RNA for interference with TMV infection utilizing a bacterial prokaryotic expression system

• Published in: Applied microbiology and biotechnology Vol. 84; no. 2; pp. 323 - 333
• Main Authors: Yin, Guohua, Sun, Zhaonan, Liu, Nan, Zhang, Lin, Song, Yunzhi, Zhu, Changxiang, Wen, Fujiang
• Format: Journal Article
• Language: English
• Published: Berlin/Heidelberg Berlin/Heidelberg : Springer-Verlag 01.08.2009; Springer Berlin Heidelberg; Springer; Springer Nature B.V
• Subjects: Applied Genetics and Molecular Biotechnology; Biological and medical sciences; Biology; Biotechnology; DNA polymerase; E coli; Escherichia coli; Escherichia coli - genetics; Escherichia coli - metabolism; Escherichia coli Proteins - genetics; Fundamental and applied biological sciences. Psychology; Gene expression; Genes; Genes, Bacterial; Genetic engineering; Genetic Vectors; Genomics; Host-Parasite Interactions - genetics; Infections; Introduced species; Laboratories; Life Sciences; Microbial Genetics and Genomics; Microbiology; Monosaccharide Transport Proteins - genetics; Mutation; Nicotiana - genetics; Nicotiana - virology; Plant Diseases - genetics; Plant Diseases - virology; Plasmids; Ribonuclease III - genetics; Ribonucleic acid; RNA; RNA Interference; RNA, Double-Stranded - biosynthesis; Studies; Symporters - genetics; Tobacco mosaic virus; Tobacco Mosaic Virus - genetics; Viruses; RNA-mediated virus resistance; TMV; Double-stranded RNA; RNase III; Infection; Resistance; Virus; Double stranded RNA; Interference; Production; Bacteria; Gene expression
• ISSN: 0175-7598; 1432-0614
• DOI: 10.1007/s00253-009-1967-y

In many species, the introduction of double-stranded RNA (dsRNA) induces potent and specific gene silencing, a phenomenon called RNA interference (RNAi). RNAi is the process of sequence-specific, posttranscriptional gene silencing (PTGS) in animals and plants, mediated by dsRNA homologous to the silenced genes. In plants, PTGS is part of a defense mechanism against virus infection, and dsRNA is the pivotal factor that induces gene silencing. Here, we report an efficient method that can produce dsRNA using a bacterial prokaryotic expression system. Using the bacteriophage λ-dependent Red recombination system, we knocked out the rnc genes of two different Escherichia coli strains and constructed three different vectors that could produce dsRNAs. This work explores the best vector/host combinations for high output of dsRNA. In the end, we found that strain M-JM109 or the M-JM109lacY mutant strain and the vector pGEM-CP480 are the best choices for producing great quantities of dsRNA. Resistance analyses and Northern blot showed that Tobacco mosaic virus infection could be inhibited by dsRNA, and the resistance was an RNA-mediated virus resistance. Our findings indicate that exogenous dsRNA could form the basis for an effective and environmentally friendly biotechnological tool that protects plants from virus infections.