A novel method for constructing pathogen-regulated small RNA cDNA library

Pathogen-responsive endogenous small non-coding RNAs regulate gene expression in relation to plant immune responses by serving as RNA silencing machinery. Decay caused by the bacterium, Erwinia carotovora subsp . carotovora ( Ecc), often leads to soft rot disease in the plant Brassica campestris L....

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Bibliographic Details
Published in:Biochemical and biophysical research communications 2010-07, Vol.397 (3), p.532-536
Main Authors: Sun, Chuan Bao, Du, Xian Ming, He, Yu Ke
Format: Article
Language:English
Subjects:
Base Sequence
Brassica - genetics
Brassica - microbiology
Brassica campestris
cDNA/RNA subtractive hybridization
Cloning, Molecular - methods
DNA, Single-Stranded - genetics
Erwinia carotovora
Gene Library
Host-Pathogen Interactions - genetics
MicroRNAs - genetics
Nucleic Acid Hybridization
Pectobacterium carotovorum - physiology
Plant Diseases - genetics
Plant Diseases - microbiology
Small non-coding RNA
Stem–loop primer
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Summary:Pathogen-responsive endogenous small non-coding RNAs regulate gene expression in relation to plant immune responses by serving as RNA silencing machinery. Decay caused by the bacterium, Erwinia carotovora subsp . carotovora ( Ecc), often leads to soft rot disease in the plant Brassica campestris L. ssp . pekinensis ( Bcp). To discover endogenous small RNA species in Bcp in response to Ecc infection, we developed a highly efficient approach for cloning pathogen-regulated small RNAs. A group of degenerate stem–loop reverse primers was designed to synthesize first single-stranded cDNA (sscDNA) and the sscDNA was then tailed with a poly(C) at its 3′ end to create a forward priming site. A novel cDNA/RNA subtractive hybridization was performed to capture Ecc-regulated small RNAs and this subsequently allowed construction of small RNA cDNA libraries for sequencing.
ISSN:0006-291X
1090-2104
DOI:10.1016/j.bbrc.2010.05.151