title: Study on Expression Modes and Cleavage Role of miR156b/c/d and its Target Gene Vv-SPL9 During the Whole Growth Stage of Grapevine.
abstract: miR156 regulates the expression of its target SPL (PROMOTER BINDING-LIKE) genes during flower and fruit development, diverse developmental stage transitions, especially from vegetative to reproductive growth phases, by cleaving the target mRNA SPL of one plant-specific transcription factor. However, systematic reports on grapevine have yet to be presented. Here, the precise sequence of miR156 (vvi-miR156b/c/d) in grapevine "Takatsuma" was cloned with a previously cloned grapevine SPL (Vv-SPL9). Expression profiles in 18 grapevine tissues were identified through stem-loop RT-PCR. The interaction mode between vvi-miR156b/c/d and Vv-SPL9 was further validated by detecting the cleavage site and cleavage products of 3'- and 5'-ends via an integrated approach of 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-PCR (real time reverse transcriptase-polymerase chain reaction). The variation in their cleavage roles in the whole growth stage of grapevine was also systematically investigated. Results showed that vvi-miR156b/c/d exhibited typical temporal-spatial-specific expression levels. The expression levels were higher in vegetative organs, such as leaf, than in reproductive organs, such as tendrils, flowers, and berries. A significant variation was observed during vegetative-to-reproductive transition. The expression patterns of Vv-SPL9 showed the opposite trends with those of vvi-miR156b. We confirmed that the cleavage site was at the 10th site of vvi-miR156b/c/d complementary to Vv-SPL9 in "Takatsuma" grapevine. We also identified the temporal-spatial variation of the cleavage products. This variation can indicate the regulatory function of miR156 on SPL in grapevines. Our findings provide further insights into the functions of vvi-miR156b/c/d and its target Vv-SPL9, and also help enrich our knowledge of small RNA-mediated regulation in grapevine.
authors.affiliation: From the College of Horticulture, Nanjing Agricultural University, Nanjing 210095, China (B. Wang, J. Wang, C. Wang, Jia, Zhu, and Li); and College of Life Science, Nanjing Agricultural University, Nanjing 210095, China (Shen).
authors.forename: Baoju
authors.initials: B
authors.lastname: Wang
authors.affiliation: From the College of Horticulture, Nanjing Agricultural University, Nanjing 210095, China (B. Wang, J. Wang, C. Wang, Jia, Zhu, and Li); and College of Life Science, Nanjing Agricultural University, Nanjing 210095, China (Shen).
authors.forename: Jian
authors.initials: J
authors.lastname: Wang
authors.affiliation: From the College of Horticulture, Nanjing Agricultural University, Nanjing 210095, China (B. Wang, J. Wang, C. Wang, Jia, Zhu, and Li); and College of Life Science, Nanjing Agricultural University, Nanjing 210095, China (Shen). wangchen@njau.edu.cn.
authors.forename: Chen
authors.initials: C
authors.lastname: Wang
authors.affiliation: From the College of Horticulture, Nanjing Agricultural University, Nanjing 210095, China (B. Wang, J. Wang, C. Wang, Jia, Zhu, and Li); and College of Life Science, Nanjing Agricultural University, Nanjing 210095, China (Shen).
authors.forename: Wenbiao
authors.initials: W
authors.lastname: Shen
authors.affiliation: From the College of Horticulture, Nanjing Agricultural University, Nanjing 210095, China (B. Wang, J. Wang, C. Wang, Jia, Zhu, and Li); and College of Life Science, Nanjing Agricultural University, Nanjing 210095, China (Shen).
authors.forename: Haifeng
authors.initials: H
authors.lastname: Jia
authors.affiliation: From the College of Horticulture, Nanjing Agricultural University, Nanjing 210095, China (B. Wang, J. Wang, C. Wang, Jia, Zhu, and Li); and College of Life Science, Nanjing Agricultural University, Nanjing 210095, China (Shen).
authors.forename: Xudong
authors.initials: X
authors.lastname: Zhu
authors.affiliation: From the College of Horticulture, Nanjing Agricultural University, Nanjing 210095, China (B. Wang, J. Wang, C. Wang, Jia, Zhu, and Li); and College of Life Science, Nanjing Agricultural University, Nanjing 210095, China (Shen).
authors.forename: Xiaopeng
authors.initials: X
authors.lastname: Li
mesh_terms: D003001:Cloning, Molecular; D020869:Gene Expression Profiling; D018507:Gene Expression Regulation, Developmental; D018506:Gene Expression Regulation, Plant; D017343:Genes, Plant; D056726:Genetic Association Studies; D035683:MicroRNAs / Q000235:genetics*; D010641:Phenotype; D034622:RNA Interference; D012333:RNA, Messenger / Q000235:genetics*; D027843:Vitis / Q000235:genetics* / Q000254:growth & development*
chemical_list: D035683:MicroRNAs; D012333:RNA, Messenger
keywords: 
  Vv-SPL9
  ; PPM-RACE; RLM-RACE; Vv-mi156; cleavage role; grapevine; qRT-PCR