Scientia Agricultura Sinica ›› 2021, Vol. 54 ›› Issue (6): 1199-1217.doi: 10.3864/j.issn.0578-1752.2021.06.011

• HORTICULTURE • Previous Articles     Next Articles

Function Analysis of vvi-miR172s and Their Target Genes Response to Gibberellin Regulation of Grape Berry Development

XuXian XUAN1(),ZiLu SHENG1,ZhenQiang XIE2,YuQing HUANG1,PeiJie GONG1,Chuan ZHANG1,Ting ZHENG1,Chen WANG1(),JingGui FANG1   

  1. 1College of Horticulture, Nanjing Agricultural University, Nanjing 210095
    2Jiangsu Vocational College of Agriculture and Forestry, Jurong 212499, Jiangsu
  • Received:2020-06-01 Accepted:2020-11-19 Online:2021-03-16 Published:2021-03-25
  • Contact: Chen WANG E-mail:2019804199@njau.edu.cn;wangchen@njau.edu.cn

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Abstract:

【Objective】 miR172 is an important regulator of plant growth and development. The purpose of this research was to clarify the roles and modes response to gibberellin (GA) of the miR172 family and its target genes in development of different tissues of grape berry. 【Method】The mature and precursor sequences of vvi-miR172a/b/c/d were cloned and identified by miR-RACE and PCR techniques from grapevine cv. Rosario Bianco. The target genes of vvi-miR172s were predicted by psRNA Target software, and the phylogenetic, structure analysis and subcellular localization were performed by bioinformatics tools. RLM-RACE verified the cleavage roles of four target genes by vvi-miR172s. The cis-elements analysis of their promoters was predicted by PLANTCARE software. The qRT-PCR method was used to detect the temporal and spatial expression patterns of vvi-miR172s and target genes in different tissues of grape berry induced by exogenous GA3 application. 【Result】The mature and precursor sequences of vvi-miR172a/b/c/d were cloned, and four target genes (VvAP2, VvRAP2-1, VvRAP2-2 and VvRAP2-3) were identified from grape genome. The cleavage sites of vvi-miR172s on target genes were detected by RLM-RACE, which proved that VvAP2, VvRAP2-1, VvRAP2-2 and VvRAP2-3 were the true target genes. Gene structure analysis result showed that all target genes contained 10 exons, 9 introns and 2 AP2 domains. The type and number of motif elements were similar, indicating that gene structures were highly conserved. Phylogenetic analysis showed that VvAP2, VvRAP2-1, and VvRAP2-2 were closer to poplar, and VvRAP2-3 had high homology with soybean. The secondary structure prediction of target proteins indicated that they existed in the form of alpha-helix and further folded into the stable tertiary structure. Subcellular localization results showed that all target proteins were mainly located in the nucleus. Both vvi-miR172c and VvRAP2-1 had the hormone related cis-elements response to GA3, suggesting that they might be involved in the regulation of grape berry development by responding to corresponding hormones. Microarray data analysis revealed that the expression patterns of vvi-miR172c and target genes were tissue or orGA3n specific. RT-qPCR analysis showed that vvi-miR172a/b/d showed a decrease expression trend in pericarp, while VvRAR2-1 exhibited a reverse expression trend to the former, indicating that vvi-miR172a/b/d neGA3tively regulated VvRAR2-1. However, in flesh, VvAP2 was contradictory to vvi-miR172d, with the increased expression during grape development, suggesting that there was a significant neGA3tive correlation between vvi-miR172d and VvAP2. Interestingly, GA3 treatment promoted the neGA3tive regulation of vvi-miR172d on VvRAP2-1 and induced the neGA3tive regulation of vvi-miR172c on VvAP2/VvRAP2-1/VvRAP2-3. 【Conclusion】VvAP2, VvRAP2-1, VvRAP2-2, and VvRAP2-3 were the real target genes of vvi-miR172s. Among the vvi-miR172 family, vvi-miR172 a/b/d might mediate VvRAP2-1 regulation of pericarp development, whereas vvi-miR172d might mediate VvAP2 involved in the development of grape flesh. vvi-miR172c/d and their target genes might participate in the regulation of grape pericarp and flesh development in response to exogenous GA.

Key words: grape, fruit development, gibberellin, miR172, target gene

Table 1

Sequence and use of primers"

引物名称 Primer name 引物序列 Primer sequence 引物用途 Primer use
vvi-miR172a-F AAGCTTTCGTAGTTGTCTACAAAATGATCTCT vvi-miR172a前体序列的扩增
Amplification of vvi-miR172a precursor
vvi-miR172a-R GGATCCCATCGCTTTACATTTTAGCCACT
vvi-miR172b-F AAGCTTCGGCATCTTTCTCCTCATACC vvi-miR172b前体序列的扩增
Amplification of vvi-miR172b precursor
vvi-miR172b-R GGATCCGGGTTGAAGACGTACATAAATCTCA
vvi-miR172c-F AAGCTTTTTGAAGCGTTCTGCTGTC vvi-miR172c前体序列的扩增
Amplification of vvi-miR172c precursor
vvi-miR172c-R GGATCCCAGATGAAACACTTCCAAGAGA
vvi-miR172d-F AAGCTTGAAGCGAGATCCGTATCCTC vvi-miR172d前体序列的扩增
Amplification of vvi-miR172d precursor
vvi-miR172d-F GGATCCATCTCAAATTACTGCATGATTTCAA
Actin-F TACAATTCCATCATGAAGTGTGATG Actin内参引物
Actin internal reference primer
Actin-R TTAGAAGCACTTCCTGTGAACAATG
VvAP2 qRT-F CTCGCGCATATGATAGGGCT VvAP2定量RT-PCR引物
Real-time PCR of VvAP2
VvAP2 qRT-R ACTTTGTCGGCGAAGGACAT
VvRAP2-1 qRT-F CAACACTGGGGGTTCATCCA VvRAP2-1定量RT-PCR引物
Real-time PCR of VvRAP2-1
VvRAP2-1 qRT-R TTTTGCCATGCCCAGTTTGG
VvRAP2-2 qRT-F CTGACTACCGTGCAAGGGTT VvRAP2-2定量RT-PCR引物
Real-time PCR of VvRAP-22
VvRAP2-2 qRT-R TTTGATCACCCTGGCCTAGC
VvRAP2-3 qRT-F GGGCTAGCAATGACATCGGA VvRAP2-3定量RT-PCR引物
Real-time PCR of VvRAP2-3
VvRAP2-3 qRT-R CATTTGCCACCCCCAGTTTG

Fig. 1

Growth and development of White Rosario grape under GA3 and CK treatments"

Fig. 2

Identification of vvi-miR172a/b/c/d precursor sequences and mature sequences alignment of miR172 in different species A: PCR products of vvi-miR172a/b/c/d primary sequences; 1: vvi-miR172a; 2: vvi-miR172b; 3: vvi-miR172c; 4: vvi-miR172d. B: Chromosomal location of vvi-miR172s and their targets genes; C: Hairpin structure of vvi-miR172s; D: Mature sequences alignment of miR172 in different species"

Table 2

Information of vvi-miR172s and target genes in grapevine"

miRNAs MiRNA成熟
体长度
Length of miRNA		 靶基因编号
Target_Acc.		 靶基因长度
Length of target		 靶区
Target regions		 靶区起始、
终止位点
Target start and stop sites		 错配率
Expectation		 允许不匹配
的最大能量
UPE		 切割方式
Interaction mode
vvi-miR172a/b 21 VIT_07s0031g00220(VvAP2) 2565 CDS:1870-1890 C、C 2.5 15.05 裂解Cleavage
21 VIT_08s0040g03180(VvRAP2-1) 1730 CDS:1331-1351 C、T 2.5 11.489 裂解Cleavage
21 VIT_13s0019g03550(VvRAP2-2) 2217 CDS:1566-1586 C、C 2.5 17.172 裂解Cleavage
21 VIT_06s0004g03590(VvRAP2-3) 2347 CDS:1806-1826 C、T 2.5 14.04 裂解Cleavage
vvi-miR172c 21 VIT_07s0031g00220(VvAP2) 2565 CDS:1870-1890 C、C 0.5 15.05 裂解Cleavage
21 VIT_08s0040g03180(VvRAP2-1) 1730 CDS:1331-1351 C、T 1.0 11.489 裂解Cleavage
21 VIT_13s0019g03550(VvRAP2-2) 2217 CDS:1566-1586 C、C 0.5 17.172 裂解Cleavage
21 VIT_06s0004g03590(VvRAP2-3) 2347 CDS:1806-1826 C、T 1.0 14.04 裂解Cleavage
vvi-miR172d 23 VIT_07s0031g00220(VvAP2) 2565 CDS:1870-1892 C、A 2.0 15.552 裂解Cleavage
23 VIT_08s0040g03180(VvRAP2-1) 1730 CDS:1331-1353 C、G 1.5 9.197 裂解Cleavage
23 VIT_13s0019g03550(VvRAP2-2) 2217 CSS:1566-1588 G、A 2.5 22.9 裂解Cleavage
23 VIT_06s0004g03590(VvRAP2-3) 2347 CDS:1806-1828 C、A 0.5 14.075 裂解Cleavage

Fig. 3

Match degree of vvi-miR172s and their target genes"

Fig. 4

The cleavage site between vvi-miR172s and target genes The dotted lines represent the cleavage sites; numbers indicate the frequency of clones at 5 termini of mRNA fragments"

Fig. 5

Phylogenetic relationship and gene structure and conserved motifs analysis of target genes A: Phylogeny tree of target genes; B: Gene structure of target genes; C: The conserved motifs of target genes"

Fig. 6

Structure analysis and subcellular localization prediction of target proteins A: The secondary structure prediction of target proteins; B: The tertiary structure prediction of target proteins; C: The conserved domains prediction of target proteins; D: The subcellular localization prediction of target proteins"

Fig. 7

Predicted cis-elements in the promoter of vvi-miR172s and their target genes A: The total number of diverse types of cis-elements derived from vvi-miR172s and their target genes promoters; B: Hormone-related cis-elements of vvi-miR172s and their target genes"

Fig. 8

Expression levels of vvi-miR172s (GEO:GSE59802) and target genes (GEO:GSE36128) in different orGA3ns, tissues and development stages A: Expression profiles of vvi-miR172c and target genes in different orGA3ns, tissures and development satges; B: Expression of vvi-miR172c and target genes in different tissues. Bud-AB: Bud after brust; Inflorescence-WD: Well development inflorescence; Flower-FB: Flowering begins; Flower-F: Flowering; Rachis-PFS: Rachis post fruit set; Stem-G: Green stem; Stem-W: Woody stem; Seed-G: Seed collected from green berries"

Fig. 9

Expression levels of vvi-miR172a/b/c/d and its target genes in berry pericarp (A) and flesh (B) of grape *,** represented significant difference (P<0.05) and extremely significant difference (P<0.01), respectively. The same as below"

Fig. 10

Expression levels of vvi-miR172a/b/c/d and target genes in berry pericarp (A) and flesh (B) of grape under GA3 treatment"

Fig. 11

Differential expression and correlation analysis of each member of vvi-miR172 family and target genes in response to GA3 treatment in berry pericarp (A) and flesh (B) of grape"

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