Scientia Agricultura Sinica ›› 2020, Vol. 53 ›› Issue (9): 1890-1903.doi: 10.3864/j.issn.0578-1752.2020.09.015

• HORTICULTURE • Previous Articles     Next Articles

vvi-miR160s in Mediating VvARF18 Response to Gibberellin Regulation of Grape Seed Development

YunHe BAI,WenRan WANG,TianYu DONG,Le GUAN,ZiWen SU,HaiFeng JIA,JingGui FANG,Chen WANG()   

  1. College of Horticulture, Nanjing Agricultural University, Nanjing 210095
  • Received:2019-09-05 Accepted:2019-12-25 Online:2020-05-01 Published:2020-05-13
  • Contact: Chen WANG E-mail:wangchen@njau.edu.cn

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Abstract:

【Objective】This study was performed to investigate the roles and the modes responsive to gibberellin (GA) of the vvi-miR160 family and its target genes in the development of Wink grape seed. 【Method】miR-RACE, RT-qPCR, bioinformatics and RLM-RACE were employed to identify vvi-miR160s and its target gene, and to analyze their modes responsive to GAof spatio-temporal expression and potential functions. 【Result】GA treatment before flowering strongly inhibited the ovule and seed development of Wink grape and induced grape seedless berries with high efficiency, and the seedless rate of berries reached 99.8%. The precursor gene sequence (501 bp) and mature sequence of vvi-miR160s were cloned and identified, which were highly conserved across different plant species. The mature sequences of vvi-miR160s were used as queries to predict the target gene VvARF18. The cleavage sites with 9/17 being their cleavage frequency of vvi-miR160s on VvARF18 were detected between the 10th and 11th sites by RLM-RACE and PPM-RACE, which proved that VvARF18 was the true target gene of vvi-miR160s. VvARF18 encoded 683 amino acids, and a nuclear localization signal existed at positions 398-411, while the protein sub-cellular was localized on the nucleus. The homology of VvARF18 with other in other species was highly conserved. The VvARF18 protein was closely related to tea, tobacco, plum and other species. The number of elements and their order were the same across different species, and the genes structures were similar. The VvARF18 promoter contained four types of cis-elements, which possessed more hormone-related cis-elements. RT-qPCR analysis showed that vvi-miR160c/d/e showed a ‘V’-shaped expression trend with the development of grape berries, and the lowest expression levels were found during the stone-hardening stage. VvARF18 exhibited an opposite expression trend to the former, with the highest expression during stone-hardening stage, indicating that vvi-miR160c/d/e negatively regulates VvARF18, but there was no significant negative correlation between vvi-miR160a/b and VvARF18 expression levels. GA treatment significantly up-regulated the expression of vvi-miR160a/b in the development of grape hardcore seeds, and also conspicuously inhibited the expression of VvARF18 in the corresponding period. The expression levels between vvi-miR160a/b and VvARF18 under GA treatments showed the typical negative correlation, indicating that GA treatment promoted the negative regulation of vvi-miR160a/b on VvARF18; reversely, GA weakened the negative regulation of vvi-miR160c/d/e on VvARF18. 【Conclusion】Among the vvi-miR160 family, vvi-miR160c/d/e may mediated VvARF18 regulation of seed development during specific stages of grape seed development, whereas vvi-miR160a/b may mediated VvARF18, which might be mainly involved in the regulation of GA-induced grape seedless berry development.

Key words: grape, vvi-miR160s, VvARF18, gibberellin (GA), seed development

Table 1

VvMIR160s PCR amplification primer sequences"

基因名称 Gene name 正向引物序列 Forward primer sequence 反向引物序列 Reverse primer sequence
VvMIR160a ACACCTCCTAAAATCATTGTCTG CTTGTGACATGAATATGGTGCG
VvMIR160b CTATGTATTTGTCTTGTTCTGATTGAA TGAATGGTCACAGTTCTTTGG
VvMIR160c GGCCTGGCCTCTATAAATATCA AATCGACCCACAATCAAACC
VvMIR160d GATGTGGTGCTTCGCCAAT ATGTGGGTTTTCTAAATGCCTAACC
VvMIR160e CACTCACTCACACCCTTCC ATATTATATTCTCTCTGCAGCCAAG

Table 2

vvi-miR160s and VvARF18 quantitative primer sequence"

基因名称 Gene name 正向引物序列 Forward primer sequence 反向引物序列 Reverse primer sequence
vvi-miR160a TGACCTTTGTGCTTCAGTGG GCTATCTGGGTTGACCTCCA
vvi-miR160b TTCTGCAGGAGATGGAGCTT AGTGTTTCGCCTGCTTGACT
vvi-miR160c CCACATTCCGTGACCTTTCT GCACAACCCATTTCACCTTT
vvi-miR160d CGCCAATGCAGGAAATTTAT GGGAGCCAGGCATGTAAGTA
vvi-miR160e CTGTATGCCATTTGCAGAGC GGGGGAGAAGATTGAAGAGG
VvARF18 CTGAACACGCCTATGGGAAT CCGTTTCACCCTCAGTGTTT

Fig. 1

Effect of GA treatment on fruit development of Wink grape"

Fig. 2

Sequence analysis and chromosome distribution of mature vvi-miR160s"

Fig. 3

MicroR160s evolution analysis and mature sequence alignment"

Fig. 4

vvi-miR160s and target gene complementary mismatch ×：Mismatch is 1, ○: Mismatch is 0.5"

Fig. 5

ARF gene sequence structure analysis"

Fig. 6

ARF18 protein evolution analysis and gene structure analysis"

Fig. 7

ARF18 protein action element"

Table 3

VvARF18 gene promoter cis-acting element"

元件类型 Component type 相关元件 Related component 数量 Number 功能注释 Functional comment
光响应元件
Photoresponsive element		 3-AF1 binding site 1 光响应元件Photoresponsive element
AE-box 1 光反应元件Photoresponsive element
Box 4 7 光响应元件Photoresponsive element
G-box 2 光响应元件Photoresponsive element
GT1-motif 6 光响应元件Photoresponsive element
激素响应元件
Hormone response element		 ABRE 2 脱落酸响应元件Abscisic acid response element
AuxRR-core 1 生长素响应元件Auxin response element
CGTCA-motif 1 茉莉酸甲酯响应元件Methyl jasmonate response element
TGACG-motif 1 茉莉酸甲酯响应元件Methyl jasmonate response element
P-box 1 赤霉素响应元件Gibberellin response element
胁迫相关元件
Stress related component		 ARE 3 厌氧诱导元件Anaerobic inducing element
LRT 1 低温诱导元件Low temperature inducing element
结合位点
Bonding component		 CCAAT-box 1 MYBHv1结合位点MYBHv1 binding site
MBS 2 MYB参与干旱诱导位点MYB participates in drought induction sites

Fig. 8

vvi-miR160s and its target gene fragmentation validation"

Fig. 9

vvi-miR160s: VvARF18 spatiotemporal expression pattern and its correlation"

Fig. 10

vvi-miR160s and VvARF18 respond to GA regulation of grapevine nuclear development"

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