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. 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.
National Key Laboratory of Plant Molecular Genetics, Shanghai Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200032, China. cbsun@sibs.ac.cn
Chuan Bao
CB
Sun
Xian Ming
XM
Du
Yu Ke
YK
He
D001483:Base Sequence; D001937:Brassica / Q000235:genetics* / Q000382:microbiology; D003001:Cloning, Molecular / Q000379:methods*; D004277:DNA, Single-Stranded / Q000235:genetics; D015723:Gene Library; D054884:Host-Pathogen Interactions / Q000235:genetics*; D035683:MicroRNAs / Q000235:genetics*; D009693:Nucleic Acid Hybridization; D016973:Pectobacterium carotovorum / Q000502:physiology*; D010935:Plant Diseases / Q000235:genetics* / Q000382:microbiology
D004277:DNA, Single-Stranded; D035683:MicroRNAs