title: Identification and characterization of microRNAs related to salt stress in broccoli, using high-throughput sequencing and bioinformatics analysis.
abstract: BACKGROUND
  MicroRNAs (miRNAs) are a new class of endogenous regulators of a broad range of physiological processes, which act by regulating gene expression post-transcriptionally. The brassica vegetable, broccoli (Brassica oleracea var. italica), is very popular with a wide range of consumers, but environmental stresses such as salinity are a problem worldwide in restricting its growth and yield. Little is known about the role of miRNAs in the response of broccoli to salt stress. In this study, broccoli subjected to salt stress and broccoli grown under control conditions were analyzed by high-throughput sequencing. Differential miRNA expression was confirmed by real-time reverse transcription polymerase chain reaction (RT-PCR). The prediction of miRNA targets was undertaken using the Kyoto Encyclopedia of Genes and Genomes (KEGG) Orthology (KO) database and Gene Ontology (GO)-enrichment analyses.
  
  
  RESULTS
  Two libraries of small (or short) RNAs (sRNAs) were constructed and sequenced by high-throughput Solexa sequencing. A total of 24,511,963 and 21,034,728 clean reads, representing 9,861,236 (40.23%) and 8,574,665 (40.76%) unique reads, were obtained for control and salt-stressed broccoli, respectively. Furthermore, 42 putative known and 39 putative candidate miRNAs that were differentially expressed between control and salt-stressed broccoli were revealed by their read counts and confirmed by the use of stem-loop real-time RT-PCR. Amongst these, the putative conserved miRNAs, miR393 and miR855, and two putative candidate miRNAs, miR3 and miR34, were the most strongly down-regulated when broccoli was salt-stressed, whereas the putative conserved miRNA, miR396a, and the putative candidate miRNA, miR37, were the most up-regulated. Finally, analysis of the predicted gene targets of miRNAs using the GO and KO databases indicated that a range of metabolic and other cellular functions known to be associated with salt stress were up-regulated in broccoli treated with salt.
  
  
  CONCLUSIONS
  A comprehensive study of broccoli miRNA in relation to salt stress has been performed. We report significant data on the miRNA profile of broccoli that will underpin further studies on stress responses in broccoli and related species. The differential regulation of miRNAs between control and salt-stressed broccoli indicates that miRNAs play an integral role in the regulation of responses to salt stress.
authors.forename: Yunhong
authors.initials: Y
authors.lastname: Tian
authors.forename: Yunming
authors.initials: Y
authors.lastname: Tian
authors.forename: Xiaojun
authors.initials: X
authors.lastname: Luo
authors.forename: Tao
authors.initials: T
authors.lastname: Zhou
authors.forename: Zuoping
authors.initials: Z
authors.lastname: Huang
authors.forename: Ying
authors.initials: Y
authors.lastname: Liu
authors.forename: Yihan
authors.initials: Y
authors.lastname: Qiu
authors.forename: Bing
authors.initials: B
authors.lastname: Hou
authors.forename: Dan
authors.initials: D
authors.lastname: Sun
authors.forename: Hongyu
authors.initials: H
authors.lastname: Deng
authors.forename: Shen
authors.initials: S
authors.lastname: Qian
authors.affiliation: Cancer Research Institute, Southern Medical University, Guangzhou, Guangdong Province, People's Republic of China. yao.kaitai@yahoo.com.cn.
authors.forename: Kaitai
authors.initials: K
authors.lastname: Yao
mesh_terms: D001483:Base Sequence; D001937:Brassica / Q000378:metabolism*; D017124:Conserved Sequence; D059014:High-Throughput Nucleotide Sequencing; D035683:MicroRNAs / Q000378:metabolism*; D054712:Salinity; D055049:Salt Tolerance; D017423:Sequence Analysis, RNA; D013312:Stress, Physiological
chemical_list: D035683:MicroRNAs