Scientia Agricultura Sinica ›› 2019, Vol. 52 ›› Issue (16): 2858-2870.doi: 10.3864/j.issn.0578-1752.2019.16.011

• HORTICULTURE • Previous Articles     Next Articles

Identification of the Target Genes of VvmiR159s and Their Regulation in Response to GA in Different Tissues of Grape Berry

ZHANG WenYing1,HAN Xu1,ZHU XuDong1,XIE ZhenQiang2,JIU SongTao3,HUANG YuQing1,JIA HaiFeng1,FANG JingGui1,WANG Chen1()   

  1. 1 College of Horticulture, Nanjing Agricultural University, Nanjing 210095
    2 Jiangsu Vocational College of Agriculture and Forestry, Jurong 20140, Jiangsu
    3 Institute of Agriculture and Biology, Shanghai Jiao Tong University, Shanghai 200240
  • Received:2019-02-18 Accepted:2019-05-16 Online:2019-08-16 Published:2019-08-21
  • Contact: Chen WANG E-mail:wangchen@njau.edu.cn

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Abstract:

【Objective】The main objective of the present research was to identify the miR159 family members and their target genes from grapevine genome, to validate the role of VvmiR159 family members and their target genes in response to exogenous GA in the development of different tissues of seedless grape berries. 【Method】The mature and precursor sequences of VvmiR159a/b/c were cloned and identified by miR-RACE and PCR techniques from grapevine cv. ‘Rosario Bianco’. The target genes of VvmiR159s were predicted by PsRNATarget software, and the phylogenetic and conserved domain analysis was performed by bioinformatics tools. The potential functions of VvmiR159s and their target genes were predicted by cis-elements analysis of their promoters. RLM-RACE and PPM-RACE verified the cleavage roles of four target genes by VvmiR159a/b/c, and the qRT-PCR method was used to detect their temporal and spatial expression patterns in different tissues of grape berry induced by exogenous GA application 【Result】The mature and precursor sequences of VvmiR159a/b/c were cloned from the ‘Rosario Bianco’ and further folded into the typical hairpin structures. Four target genes (VvGAMYB, VvMYB48, VvAIX15, and VvGRAS-1) of VvmiR159s were identified, of which VvmiR159s strongly matched with VvGAMYB the most and VvAIX15 was weak. Phylogenetic evolution and conserved domain analysis of target genes showed that the four target genes had high homology with other species. All VvmiR159s and four target genes had the main hormone related cis-elements responsive to GA and SA, indicating that they might be involved in the regulation of grape berry growth and development mainly by responding to corresponding hormones. The expression of VvmiR159s in different tissues was spatial-temporal-specific. The expression of VvmiR159c was deviated in berry pericarp and flesh, and the expression level of VvmiR159a/b/c was negatively correlated with VvAIX15 in the pericarp, however in flesh, VvmiR159a/b was contradictory to VvGAMYB and VvAIX15 expression pattern. In addition, GA treatment VvmiR159a/c was significantly down-regulated in grape pericarp and flesh, but VvmiR159b up-regulated in young fruit flesh. These results indicated that different members of the VvmiR159 family participated in the regulation of fruit development through different modes of response to GA signaling in different tissues of the fruit. 【Conclusion】The grape miR159 family contains three members of VvmiR159a/b/c; all of three members can cleave four target genes: VvGAMYB, VvMYB48, VvAIX15 and VvGRAS-l; VvmiR159a/b/c and their four target genes may participate in the regulation of grape pericarp and flesh development in different GA-responsive modes.

Key words: grape, miR159, target gene, GA, evolutionary characteristic, gene expression

Table 1

Sequence and use of primers"

引物名称 Primer name 引物序列 Primer Sequence 引物用途 Use
VvmiR159a-F CCATGGAATCTTTTTCCGGACACTACAACA VvmiR159a前体序列的扩增
Amplification of VvmiR159a precursor
VvmiR159a-R ACTAGTATCCCACCCGGATTTTGAAATAA
VvmiR159b-F CCATGGTGTTATTGGGTTTGGGTCATCTCA VvmiR159b前体序列的扩增
Amplification of VvmiR159b precursor
VvmiR159b-R ACTAGTACCCAGATTAACAATACAAAAGGTC
VvmiR159c-F CCATGGCAGTCAGTAATGCAAGCCACA VvmiR159c前体序列的扩增
Amplification of VvmiR159c precursor
VvmiR159c-R ACTAGTAGCTGCAGTTTTGTTGGGTTT
Actin-F TACAATTCCATCATGAAGTGTGATG Actin 内参引物
Actin primer
Actin-R TTAGAAGCACTTCCTGTGAACAATG
VvmiR159a qRT-F CTTCTCCGAGGTCATGCACA VvmiR159a 定量RT-PCR引物
Real-time PCR of VvmiR159a
VvmiR159a qRT-R ATGGGAGCTCCTTTACGCTC
VvmiR159b qRT-F TTGCCATTCCAACACTCATC VvmiR159b 定量RT-PCR引物
Real-time PCR of VvmiR159b
VvmiR159b qRT-R TTTCTGGCCTTGGAGTGAAG
VvmiR159c qRT-F GCTCCCTTCAATCCAAACAA VvmiR159c 定量RT-PCR引物
Real-time PCR of VvmiR159c
VvmiR159c qRT-R TTATGGATCCCACAGCCCTA
VvGAMYB qRT-F GAAATGGAATCCCCCTGTTT VvGAMYB 定量RT-PCR引物
Real-time PCR of VvGAMYB
VvGAMYB qRT-R TCCTTGTTGGTAGGGTCAGG
VvMYB48 qRT-F TTCTCAGGAACTGGGGAATG VvMYB48 定量RT-PCR引物
Real-time PCR of VvMYB48
VvMYB48 qRT-R AAAGAAGCTGTCCGGAGTCA
VvAIX15 qRT-F GTGCCCAAAGGCTATGTTCC VvAIX15 定量RT-PCR引物
Real-time PCR of VvAIX15
VvAIX15 qRT-R TCTTCCCTGCATGGGATTGT
VvGRAS-l qRT-F TTGGAGGCCTTAGAAGGGGA VvGRAS-l 定量RT-PCR引物
Real-time PCR of VvGRAS-l
VvGRAS-l qRT-R ACAGTGAGCTTTAGGCGAGG

Fig. 1

Effects of GA treatment on fruit development of Vitis vinifera cv Rosario Bianco grapevine 10DAF: 10 days after flowering; 35DAF: 35 days after flowering; 60DAF: 60 days after flowering; 85DAF: 85 days after flowering"

Fig. 2

Identification of VvmiR159a/b/c mature and precursor sequences A: The PCR products of 3’-miR-RACE and 5’-miR-RACE for VvmiR159a/b/c, respectively; B: PCR products of VvmiR159a/b/c primary sequences; C: Comparison of cloned VvmiR159s precise sequences with the homologous sequences in grapes from MiRBase; D: Hairpin structures of VvmiR159s"

Table 2

Prediction the targets of VvmiR159a/b/c"

基因名称
Gene name		 靶基因编号
Target Acc.		 靶区
Target regions		 靶片段
Target aligned fragment		 作用方式
Interaction mode		 注释
Annotation
VvGAMYB VIT_213s0067g01630 CDS:956-976 TGGAGCTCCCTTCACTCCAAT Cleavage GAMYB转录因子
Transcription factor GAMYB
VvMYB48 VIT_215s0021g02040 CDS:97-117 TGGAGCTCCATTCGCTCCAAA Cleavage MYB48转录因子
Transcription factor MYB48
VvAIX15 VIT_203s0038g01150 CDS:152-172 TGAAGCACCCTTCATTCCAAA Cleavage 生长素诱导蛋白X15
Auxin-induced protein X15
VvGRAS-l VIT_200s0226g00070 CDS:6-26 CAGAACTCTCTTCACTCCAAA Cleavage GRAS家族转录因子
GRAS family transcription factor

Fig. 3

Match degree of VvmiR159s and their target genes"

Fig. 4

Phylogenetic analysis of target genes"

Fig. 5

Conserved motif elements analysis of target proteins"

Fig. 6

Promoter motif elements classification of VvmiR159a/b/c and their target genes A: The total number of diverse types of cis-elements derived from VvmiR159s and their four target gene promoters. B: Hormone-related cis-elements of VvmiR159s and their four target genes"

Fig. 7

Mapping of VvGAMYB/VvMYB48/VvAIX15/VvGRAS-1 cleavage sites by VvmiR159s The arrows indicate the cleavage sites. Numbers indicate the frequency of the cleavage fragments expected for cloned PCR products at the different positions"

Fig. 8

Expression of VvmiR159a/b/c and their target genes in different organizations of grapevine * represented significant difference (P<0.05). The same as below"

Fig. 9

Expression patterns of VvmiR159s and their target genes in response to GA during grape fruit development A：果皮Berry pericarp;B：果肉Berry flesh"

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