Scientia Agricultura Sinica ›› 2021, Vol. 54 ›› Issue (2): 357-369.doi: 10.3864/j.issn.0578-1752.2021.02.011

• HORTICULTURE • Previous Articles     Next Articles

Function Analysis of GA3 Mediate miR171s and Its Target Genes VvSCLs in Grape Seed Development

WANG WenRan1(),XIE ZhenQiang2,ZHUGE YaXian1,BAI YunHe1,GUAN Le1,WU WeiMin3,ZHANG PeiAn1,ZHENG Ting1,FANG JingGui1,WANG Chen1()   

  1. 1College of Horticulture, Nanjing Agricultural University, Nanjing 210095
    2Jiangsu Vocational College of Agriculture and Forestry, Zhenjiang 212499, Jiangsu
    3Insititute of Horticulture, Jiangsu Academy of Agricultural Sciences, Nanjing 210014
  • Received:2020-04-27 Accepted:2020-05-27 Online:2021-01-16 Published:2021-02-03
  • Contact: Chen WANG E-mail:2017104029@njau.edu.cn;wangchen@njau.edu.cn

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Abstract:

【Objective】The main objective of the present research was to identify the vvi-miR171s and their target genes VvSCL6/15/22 from grapevine genome, and to confirm the role of vvi-miR171s and VvSCL6/15/22 in grape seed development process and their expression pattern in response to exogenous GA3. 【Method】The mature sequences of vvi-miR171a/b/e/f/g/h were validated by miR-RACE technique from grape (Vitis vinifera L.) cv ‘Wink’. The potential target genes of vvi-miR171s were predicted by PsRNATarget software, and the chromosome localization, phylogenetic, gene structure, conserved domain and motifs analysis were performed by bioinformatics tools. The potential functions of vvi-miR171s and their target genes were predicted by promoters cis-elements analysis. RLM-RACE and PPM-RACE verified the cleavage roles of three target genes by vvi-miR171a/b/e/f/g/h, and the qRT-PCR method was used to detect their temporal expression patterns in grape seed and seedless grape induced by exogenous GA3. 【Result】Six vvi-miR171s mature sequences in grape were determined and cloned, which were consistent of the sequences of miRBase. Based on this result, the target genes of them, including VvSCL6, VvSCL15 and VvSCL22, were prophesied. The target genes bioinformatics analysis showed evolutionary genetic distance between VvSCL6/22 genes and homologous genes in apple (Monodelphis domestica), peach (Prunus persica), and cherry (Prunus avium) is relatively closed, while VvSCL15 had the same genetic distance from the homologous genes in Arabidopsis, apple, peach and cherry. Meanwhile, the gene structure of VvSCLs was similar to their close genes in phylogenetic tree. All SCL protein sequences had GRAS domain, both element types, arrangement order of motifs. And 5 same motifs for GRAS domain were identified, indicating that the SCL protein structure was relatively conservative, with the conserved functionality. Subsequently, an array of gibberellin and Salicylic acid responsive cis-acting elements were identified in vvi-miR171s and target genes VvSCLs promoter sequences, indicating their possible involvement in the regulation of GA-responsive and SA-responsive grape growth and development. Moreover, the qRT-PCR analysis revealed that the expression of vvi-miR171a was significantly repressed by GA3 in seed, while its target gene VvSCLs showed reciprocal expression pattern, particularly. There was a strong negative correlation between vvi-miR171a -VvSCL15 in the grape seed of CK and seed region of GA3 treatment. Taken together, vvi-miR171a might the major member of vvi-miR171s in response to GA3, additionally, it might involve in the grape berry seed development through negative regulating of VvSCL15 gene developmental in response to GA3. 【Conclusion】vvi-miR171a/b/e/f/g/h were identified from grapevine ‘Wink’, and all of members could cleave three target genes VvSCL6/15/22. The vvi-miR171a-VvSCL15 might be the major member in response to gibberellin and involve in regulating grape seed development.

Key words: grape, vvi-miR171s, VvSCLs, seed development, gibberellin

Fig. 1

Effect of GA3 on grape berry seed development"

Fig. 2

PCR products of vvi-miR171s in grape (a), comparison of cloned vvi-miR171s mature sequences with the homologous sequences in grapes from miRBase (b)"

Fig. 3

Phylogeny tree of miR171 family mature sequences (Dot marks denote vvi-miR171s)"

Table 1

vvi-miR171s target genes prediction results"

miRNA成员
miRNA_Acc.		 miRNA 成熟体序列
miRNA_mature sequence		 靶基因基因编号
Target_Acc.		 期望值
Expectation		 未配对时
的能量UPE		 裂解区域
Target region		 抑制作用
Inhibition		 多重性
Multiplicity
vvi-miR171a TGATTGAGCCGTGC
CAATATC		 VIT_204s0023g01380.1(VvSCL15) 0.5 -1 CDS:656-676 裂解 Cleavage 1
VIT_215s0048g00270.1 (VvSCL6) 0.5 -1 CDS:1594-1614 裂解 Cleavage 1
VIT_202s0154g00400.1 (VvSCL22) 0.5 -1 CDS:1724-1744 裂解 Cleavage 1
vvi-miR171b TGATTGAGCCGCGT
CAATATC		 VIT_204s0023g01380.1 (VvSCL15) 0.5 -1 CDS:656-676 裂解 Cleavage 1
VIT_215s0048g00270.1 (VvSCL6) 0.5 -1 CDS:1594-1614 裂解 Cleavage 1
VIT_202s0154g00400.1 (VvSCL22) 0.5 -1 CDS:1724-1744 裂解 Cleavage 1
vvi-miR171e TGATTGAGCCGCGC
CAATATC		 VIT_204s0023g01380.1 (VvSCL15) 0 -1 CDS:656-676 裂解 Cleavage 1
VIT_215s0048g00270.1 (VvSCL6) 0 -1 CDS:1594-1614 裂解 Cleavage 1
VIT_202s0154g00400.1 (VvSCL22) 0 -1 CDS:1724-1744 裂解 Cleavage 1
vvi-miR171f TTGAGCCGCGCCAA
TATCACT		 VIT_215s0048g00270.1 (VvSCL6) 1 -1 CDS:1591-1611 裂解 Cleavage 1
VIT_202s0154g00400.1 (VvSCL22) 1 -1 CDS:1721-1741 裂解 Cleavage 1
VIT_204s0023g01380.1 (VvSCL15) 1 -1 CDS:653-673 裂解 Cleavage 1
vvi-miR171g TTGAGCCGAACCAA
TATCACC		 VIT_204s0023g01380.1 (VvSCL15) 4 -1 CDS:653-673 翻译 Translation 1
VIT_215s0048g00270.1 (VvSCL6) 4 -1 CDS:1591-1611 翻译 Translation 1
VIT_202s0154g00400.1 (VvSCL22) 4 -1 CDS:1721-1741 翻译 Translation 1
vvi-miR171h TGGTTGAGCCGCGC
CAATATC		 VIT_204s0023g01380.1 (VvSCL15) 0.5 -1 CDS:656-676 裂解 Cleavage 1
VIT_215s0048g00270.1 (VvSCL6) 0.5 -1 CDS:1594-1614 裂解 Cleavage 1
VIT_202s0154g00400.1 (VvSCL22) 0.5 -1 CDS:1724-1744 裂解 Cleavage 1

Fig. 4

The diagram of cleavage site between vvi-miR171s and its targets"

Fig. 5

The distribution of vvi-miR171s and VvSCLs on grapevine chromosomes, and analysis of SCLs, motif and domain a: The distribution of vvi-miR171s and VvSCLs on grapevine chromosomes; b: The unrooted tree of SCLs protein of 5 species was generated by neighbor-joining method; c: The exon-intron composition of SCL genes; d: The conserved motifs of SCL proteins; e: Conserved domains; f: SCLs protein has common conserved motifs of the conserved domain"

Fig. 6

Motif analysis of the promoters from the vvi-miR171s precursor and their target VvSCL genes a: The total number of diverse types of motifs derived from vvi-miR171s and VvSCLs promoters; b: The proportion of each type of hormones related elements of vvi-miR171s and its targeted genes VvSCLs, respectively"

Fig. 7

Spatio-temporal expression analysis of vvi-miR171s in grape seed/seed region (a), expression pattern in response to GA3 (b) and action pattern of vvi-miR171s on target gene (c)"

Fig. 8

Total expression of vvi-miR171s and their VvSCL target genes during seed/seed region development and their patterns in response to GA3"

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