Journal of Plant Physiology 170 (2013) 943–957 Contents lists available at SciVerse ScienceDirect Journal of Plant Physiology j o ur na l ho me page: www.elsevier.com/locate/jplph Molecular biology Characterization of target mRNAs for grapevine microRNAs with an integrated strategy of modified RLM-RACE, newly developed PPM-RACE and qPCRs Chen Wang a , Jian Han a , Nicholas Kibet Korir a , Xicheng Wang b , Hong Liu b , Xiaoying Li c , Xiangpeng Leng a , Jinggui Fang a,∗ a College of Horticulture, Nanjing Agricultural University, Nanjing 210095, China b Institute of Horticulture, Jiangsu Academy of Agricultural Sciences, Nanjing 210014, China c Institute of Horticulture, Zhejiang Academy of Agricultural Sciences, Hangzhou 310021, China a r t i c l e i n f o Article history: Received 24 August 2012 Received in revised form 7 February 2013 Accepted 8 February 2013 Available online 12 April 2013 Keywords: Table grapevine MicroRNA Target mRNA 3 ′ -PPM-RACE and 5 ′ -RLM-RACE qPCR a b s t r a c t MicroRNAs (miRNAs) regulate target gene expression by mediating target gene cleavage or inhibition of translation at transcriptional and post-transcriptional levels in higher plants. Until now, many grapevine microRNAs (Vv-miRNAs) have been identified and quite a number of miRNA target genes were also ver- ified by various analysis. However, global interaction of miRNAs with their target genes still remained to perform more research. We reported experimental validation of a number of miRNA target genes in table grapevine that had been previously identified by bioinformatics in our earlier studies. To verify more predicted target genes of Vv-miRNAs and elucidate the modes by which these Vv-miRNAs work on their target genes, 31 unverified potential target genes for 18 Vv-miRNAs were experimentally ver- ified by a new integrated strategy employing a modified 5 ′ -RLM-RACE (RNA ligase-mediated 5 ′ rapid amplification of cDNA ends), 3 ′ -PPM-RACE (poly(A) polymerase-mediated 3 ′ rapid amplification of cDNA ends) and qRT-PCRs of cleavage products. The results showed that these Vv-miRNAs negatively regu- lated expression of their target messenger RNAs (mRNAs) through guiding corresponding target mRNA cleavage, of which about 94.4% Vv-miRNAs cleaved their target mRNAs mainly at the tenth nucleotide of 5 ′ -end of miRNAs. Expression levels of both miRNAs and their target mRNAs in eight tissues exhibited inverse relationships, and expressions both of cleaved targets and miRNAs indicated a cleavage mode of Vv-miRNAs on their target genes. Our results confirm the importance of Vv-miRNAs in grapevine growth and development, and suggest more study on Vv-miRNAs and targets can enrich the knowledge of miRNA mediated-regulation in grapevine. © 2013 Elsevier GmbH. All rights reserved. Introduction MicroRNAs (miRNAs) are a class of 19–24 nucleotide (nt) non- coding small RNAs, which play important regulatory roles in gene expression through mediating target messenger RNAs (mRNAs) cleavage or by repressing translation of target mRNAs in most eukaryotes (Bartel and Bartel, 2003; Carrington and Ambros, 2003; Hunter and Poethig, 2003; Allison and Vaucheret, 2004; Bartel, 2004; Allison and Bouché, 2008). An increasing number of stud- ies confirm that plant miRNAs control plant growth processes Abbreviations: cDNA, complementary DNA; EST, expressed sequence tag; miRNA, microRNA; mRNA, messenger RNA; PCR, ploymerase chain reaction; qPCR, quantitative RT-PCR; RT-PCR, reverse transcription-ploymerase chain reaction; siR- NAs, small interfering RNA; Vv-miRNA, grapevine microRNA; 3 ′ -PPM-RACE, poly(A) polymerase-mediated 3 ′ rapid amplification of cDNA ends; 5 ′ -RLM-RACE, RNA ligase-mediated 5 ′ rapid amplification of cDNA ends. ∗ Corresponding author. E-mail address: fanggg@njau.edu.cn (J. Fang). such as leaf organ morphogenesis and polarity (Emery et al., 2003; Allison et al., 2004a; Juarez et al., 2004), floral differentia- tion (Aukerman and Sakai, 2003), and boundary formation/organ separation (Allison et al., 2004b; Laufs et al., 2004), hormone signaling like auxin signaling (Allison et al., 2005), biotic and abi- otic stress response (Sunkar and Zhu, 2004; Phillips et al., 2007; Lu et al., 2008; Jagadeeswaran et al., 2009), and miRNA biogene- sis (Kurihara and Watanabe, 2004; Chen, 2005) through negatively regulating expression of corresponding target genes (Bartel, 2004; Jones-Rhoades et al., 2006). Consequently, identification and char- acterization of target mRNAs for different miRNAs in plants is essential in the analysis of miRNA functions. For this exercise, bioin- formatics prediction, cloning, deep sequencing, 5 ′ -RACE, and RNA blotting have been popular methods employed. In relation to this, a number of potential target genes for dozens of miRNAs from table grapevine microRNA (Vv-miRNAs) had been identified by bioinfor- matics analysis in our earlier work (Wang et al., 2011a). Using the newly available complete grapevine genome and large numbers of expressed sequence tag (EST) sequences, we revealed that there 0176-1617/$ – see front matter © 2013 Elsevier GmbH. All rights reserved. http://dx.doi.org/10.1016/j.jplph.2013.02.005