Scientia Agricultura Sinica ›› 2023, Vol. 56 ›› Issue (10): 1966-1981.doi: 10.3864/j.issn.0578-1752.2023.10.012

• HORTICULTURE • Previous Articles     Next Articles

Identification of the VvmiR164s-VvNAC100 Action Module and Analysis of Their Expressions Responsive to Exogenous GA During Grape Ovary Development

WANG Fei1(), XIAO YingKe1, XUAN XuXian1, ZHANG XiaoWen1, LIU Fei1, ZHA ZiXian1, DAI MengTong1, WANG XiCheng2, WU WeiMin2, FANG JingGui1, WANG Chen1()   

  1. 1 School of Horticulture, Nanjing Agricultural University, Nanjing 210095
    2 Jiangsu Academy of Agricultural Sciences, Nanjing 210014
  • Received:2022-07-04 Accepted:2022-12-27 Online:2023-05-16 Published:2023-05-17

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Abstract:

【Objective】The aim of this study was to identify grapevine miR164s (VvmiR164a/b/c/d) and their target genes, and to elucidate the regulatory roles of VvmiR164s and their target genes during exogenous GA-induced grape parthenocarpic process.【Method】Using ‘Wink’ grapes (Vitis vinifera L. Wink) as the test material, miR-RACE, PCR, RLM-RACE and PPM-RACE, qRT-PCR and bioinformatics were used to analyze the spatio-temporal expression of VvmiR164s-VvNAC100 module in response to exogenous GA and its potential functions during grape parthenocarpic process.【Result】Gibberellin application on ‘Wink’ grapes before flowering induced parthenocarpy, leading to seedless berries. The precise sequence of VvmiR164a/b/c/d was cloned and identified, and four of its target genes were predicted, including VvNAC100-1, VvNAC100-2, VvNAC098, and VvNAC021. Combining the degree of match with the comprehensive analysis of the previous data, this work focused on the analysis of the target gene VvNAC100-2, which was named as VvNAC100. The VvNAC100 cleavage site was located at position 9 and position 11 of the 5′ end of miRNA, with a cleavage frequency of 17/20 and 11/20, respectively, and was localized on Chr14, encoding 363 amino acids and containing a NAM. The VvNAC100 protein is highly conserved in amino acid sequence and functionally similar to other species, among which it is more closely related to species such as pepper and tobacco. The promoters of VvMIR164a/b/c/d and its target gene VvNAC100 both contain multiple hormone-acting elements, suggesting that it might be involved in the regulation of grape growth and development by responding to mult-hormones. The RT-qPCR results showed that the expression level of VvmiR164b tended to decrease as the grape ovary developed, while its target gene VvNAC100 showed an increasing expression trend in the early stage of ovary development with a certain negative correlation. On the other hand, VvmiR164a/c/d showed a similar expression pattern with VvNAC100 with a certain positive correlation. However, after GA treatment, the expression of VvmiR164a/c/d was highly significantly increased during parthenocarpy in the grape ovary, which also significantly suppressed the expression of VvNAC100 during this period, promoting a negative correlation between VvmiR164a/c/d-VvNAC100 their expression levels. VvmiR164b showed a decreasing trend after GA treatment, and it’s the expression levels showed some positive correlation with those of VvNAC100, indicating that GA treatment promoted the negative regulation of VvmiR164a/c/d on VvNAC100, but repressed the negative regulation of VvmiR164b on VvNAC100.【Conclusion】VvNAC100 was the true target gene of VvmiR164 family. During grape parthenocarpy, GA induced the negative regulation of VvmiR164a/c/d on the target gene VvNAC100 and suppressed that of VvmiR164b on the target gene VvNAC100. VvmiR164a/c/d were the main effectors of the VvmiR164 family involved in modulation of GA-induced grape parthenocarpy.

Key words: grape, gibberellin, parthenocarpy, VvmiR164, VvNAC

Table 1

VvmiR164s 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		 用途
Application
VvmiR164aQT GATGAATTGCCTACAGTTACCAG CCAAGGCCTCCTGCAATTT VvmiR164a前体序列的扩增
Amplification of VvmiR164a precursor
VvmiR164bQT GATGGTCAGCTGCCTAATATTG ACACGCATCTTGGATTTGCT VvmiR164b前体序列的扩增
Amplification of VvmiR164b
precursor
VvmiR164cQT CTTCATTTGCCGCTTTCG CTCCACCAAGAAAATGATAGGC VvmiR164c前体序列的扩增
Amplification of VvmiR164c
precursor
VvmiR164dQT GCAATCACTTGGGAGTTGG ATAGCCAATAGAGGGGAAAAGG VvmiR164d前体序列的扩增
Amplification of VvmiR164d
precursor
VvNAC100 ATGGAAAAGGCTCCTGACCA GGAATGTTTCCCTTCTAATCTGT VvNAC100靶全长序列的扩增
Amplification of full-length sequences of VvNAC100
VvNAC100p ATGGAAAAGGCTCCTGACC GTAATTCCAGAGGCAATCAATG VvNAC100靶片段序列的扩增
Amplification of VvNAC100 target fragment sequences
VvmiR164a-qRT TCAAAACCCGAAACAAGTCC ACCCACCCTCAACCATACAA VvmiR164a定量RT-PCR引物
Real-time PCR of VvmiR164a
VvmiR164b-qRT CGAGTGTTGAGCAAGATGGA CTCCGTGATAATTGGGGAGA VvmiR164b定量RT-PCR引物
Real-time PCR of VvmiR164b
VvmiR164c-qRT TTGGGGGTGTGAAGAAGAAG GAGGAACCCATGTTGGAGAA VvmiR164c 定量RT-PCR引物
Real-time PCR of VvmiR164c
VvmiR164d-qRT TTGGGGGTGTGAAGAAGAAG GAGGAACCCATGTTGGAGAA VvmiR164d定量RT-PCR引物
Real-time PCR of VvmiR164d
VvNAC100-qRT TTCTCATCCCTGACCAGTCC TCTACAGGCCCAGCTGAAGT VvNAC100定量RT-PCR引物
Real-time PCR of VvNAC100
Vvactin GCTCGCTGTTTTGCAGTTCTAC AACATAGGTGAGGCCGCACTT 葡萄actin内参基因
Actin internal reference primer

Fig. 1

Effects of GA treatment on parthenogenesis of Wink grape"

Fig. 2

Sequence analysis and chromosomal distribution of VvmiR164s A: Amplification of precursor genes of VvmiR164s; B: Comparison of mature body sequences of VvmiR164 family; C: VvmiR164a/b/c/d chromosome localization and stem-loop structure"

Fig. 3

Evolutionary analysis (A) and mature sequence alignment (B) of VvmiR164s"

Table 2

Information about grape VvmiR164s and target genes"

miRNAs MiRNA
成熟体长度
Length of miRNA		 靶基因编号
Target_Acc.		 错配率Expectation 靶区
Target region		 靶基因匹配
初始位置
Target_start		 靶基因匹配
初始位置
Target_end		 切割方式
Interaction
mode inhibition
VvmiR164a/c/d
(UGGAGAAGCAGGGCACGUGCA)		 21 VIT_217s0000g06400 (VvNAC100-1) 0.5 gene=VIT_217s0000g06400 CDS=732-1814 1375 1395 裂解 Cleavage
21 VIT_219s0014g02200 (VvNAC098) 1.5 gene=VIT_219s0014g02200 CDS=1105-2196 1826 1846 裂解 Cleavage
21 VIT_219s0027g00230 (VvNAC021) 1.8 gene=VIT_219s0027g00230 CDS=317-1219 939 959 裂解 Cleavage
21 VIT_214s0108g01070(VvNAC100-2) 1.5 gene=VIT_214s0108g01070 CDS=348-1439 988 1008 裂解 Cleavage

VvmiR164b
(UGGAGAAGCAGGGCACAUGCU)		 21 VIT_217s0000g06400(VvNAC100-1) 1.0 gene=VIT_217s0000g06400 CDS=732-1814 1375 1395 裂解 Cleavage
21 VIT_219s0014g02200 (VvNAC098) 2.5 gene=VIT_219s0014g02200 CDS=1105-2196 1826 1846 裂解 Cleavage
21 VIT_219s0027g00230 (VvNAC021) 1.8 gene=VIT_219s0027g00230 CDS=317-1219 939 959 裂解 Cleavage
21 VIT_214s0108g01070(VvNAC100-2) 2.5 gene=VIT_214s0108g01070 CDS=348-1439 988 1008 裂解 Cleavage

Fig. 4

The degree of matching between VvmiR164s and target genes"

Fig. 5

Clonal identification, subcellular localization and sequence analysis of VvNAC100 A: Chromosomal localization of VvNAC100 gene; B: Cloning of VvNAC100 gene; C: Subcellular localization of VvNAC100 gene; D: Structural domain analysis of VvNAC100"

Fig. 6

NAC protein evolution analysis (A), conserved motif analysis (B) and element analysis (C)"

Fig. 7

Verification of VvmiR164s and its target gene cleavage A: Schematic diagram of cleavage sites of VvmiR164s on target gene VvNAC100; B: Schematic diagram of vector construction; C: GUS staining of tobacco cotransformed leaves"

Fig. 8

Promoter cis-acting elements of VvmiR164s and VvNAC100"

Fig. 9

Patterns of VvmiR164s and VvNAC100 during grape ovary development"

Fig. 10

The pattern of VvmiR164s and VvNAC100 in response to gibberellin during grape flower development"

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