Scientia Agricultura Sinica ›› 2021, Vol. 54 ›› Issue (13): 2843-2857.doi: 10.3864/j.issn.0578-1752.2021.13.013

• HORTICULTURE • Previous Articles     Next Articles

Isolation of PmARF17 and Its Regulation Pattern of Endogenous Hormones During Flower Development in Prunus mume

LI YanLin1(),SHAHID Iqbal1,SHI Ting1,SONG Juan2,NI ZhaoJun1,GAO ZhiHong1()   

  1. 1College of Horticulture, Nanjing Agricultural University, Nanjing 210095
    2Jiangsu Academy of Agricultural Sciences, Nanjing 210014
  • Received:2020-08-27 Revised:2020-10-14 Online:2021-07-01 Published:2021-07-12
  • Contact: ZhiHong GAO E-mail:lisaltp@qq.com;gaozhihong@njau.edu.cn

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Abstract:

【Objective】The purposes of this study were to analyze the biological functions of PmARF17gene and to explore the regulation pattern between the expression of PmARF17 and plant endogenous hormones, which could provide the basis for studying the regulatory mechanism of pistil abortion inPrunus mume. 【Method】 PmARF17 gene was isolated from the cultivar Daqiandi of Prunus mume, and the gene structure, phylogeny and homology with other species were analyzed by bioinformatics software. Subcellular localization determined the position of PmARF17 protein in the cell. qRT-PCR was used to detect the spatiotemporal expression pattern of PmARF17in different stages of flower buds, leaf buds, flower buds with different pistil morphology, and different mature floral organs of the varieties Daqiandi and Longyan. The contents of IAA, GA3, ABA and ZT in flower buds, leaf buds, pistil morphology flower buds and different mature flower organs of Daqiandi and Longyan were determined by UPLC method, and the correlation analysis was conducted with the qRT-PCR expression ofPmARF17.Cloning, element analysis and transient expression of PmARF17 promoter were performed to study the regulation pattern between PmARF17 and GA3. 【Result】Phylogenetic tree analysis showed PmARF17 protein sequence was highly conserved with ARF protein of other plants. Subcellular localization experiment showed PmARF17 protein was located on the nucleus and cell membrane. The correlation analysis between qRT-PCR expression and endogenous hormone content showed the expression of PmARF17 had no obvious similarity with the change trend of IAA content in different flower samples. The expression of PmARF17 in complete pistil was significantly up-regulated compared to incomplete pistil, and GA3 content was consistent with the expression trend ofPmARF17. The content of ABA and ZT in different flower samples and the expression of PmARF17 showed an overall opposite trend, indicating that they might inhibit the expression of PmARF17. The PmARF17promoter contained GAcis-element and had promoter activity and tissue expression specificity, specifically expressed in petals, stamens and roots. 【Conclusion】PmARF17 might be a positive regulation gene of flower development, and it might promote pistil development of Prunus mume. The expression of PmARF17 might be positively regulated by GA3, which affected the pistil development of Prunus mume by acting on stamens and petals.

Key words: Prunus mume, auxin response factor, pistil development, gene expression, endogenous hormones, transient expression

Table 1

Sequence and use of primers"

引物名称Primer name 引物序列 Primer Sequence (5′-3′) 引物用途 Primer use
PmARF17gene-F GCAGACATGAAACCTCCTCCTATGC PmARF17基因扩增
Amplification of PmARF17 gene
PmARF17gene-R CAAGCCATTGTATCATTGCTATCC
PmARF17 GFP-F GGAAGATCTATGGGGGGTCTAATCGATC 亚细胞定位
Subcellular localization
PmARF17 GFP-R GGAAGATCTTCCTTGTAAGCAGCTTTCC
PmARF17qPCR-F TTAGTGGCAGTCAGGATG 实时荧光定量PCR
qRT-PCR
PmARF17qPCR-R GCAGTTGAGGTTGAGTTG
Actin-F TGAAGCATACACCTATGATGATGAAG 内参引物
Actin primer
Actin-R CTTTGACAGCACCAGTAGATTCC
PmARF17 promoter-F GAGATGCCTAACACTCTAAAGTC PmARF17启动子扩增
Amplification of PmARF17promoter
PmARF17 promoter-R CCTCCGTTGCACTGTTCAGATCG
Promoter-ApaI-F GGGGCCCGAGATGCCTAACACTCTAAAGTC 瞬时表达试验
Transient expression
Promoter-SmaI-R CCCCCGGGGTGGAAATAAACAAAAATTGTA

Fig. 3

Structure of pCAMBIA1302-PmARF17-GFP vector and the subcellular localization of PmARF17 protein A: Structure of pCAMBIA1302-PmARF17-GFP recombinant vector; B: pCAMBIA1302-GFP alone; C: fusion plasmid pCAMBIA1302-PmARF17-GFP. Images under blight field (middle), fluorescence (left) and the merged images were shown on the right (bars=20 000 nm) "

Fig. 1

The gene structure of PmARF17and the phylogenetic tree and homology comparison of PmARF17 protein with other plants A: cDNA and deduced amino acid sequence of PmARF17 (The maximum ORF was underlined and the shaded areas were conservative); B: Phylogenetic tree of PmARF17 protein with other plants; C: The gene structure of PmARF17; D: Homology comparison of PmARF17 protein with other plants [The blue boxes are B3 DNA binding domain, red boxes are Auxin response factor. 1.seq: Prunus mume; 2.seq: Prunus sibirica ; 3.seq: Prunus persica ; 4.seq: Prunus avium ; 5.seq: Malus domestica; 6.seq: Pyrus x bretschneideri ; 7.seq: Vitis vinifera; 8.seq: Fragaria vesca; 9.seq: Populus trichocarpa; 10.seq: A. thaliana; 11.seq: N. tabacum ; 12.seq: Solanum lycopersicum]"

Fig. 2

Analysis of cis-acting elements of PmARF17 promoter A: Plant hormone response elements of PmARF17 promoter; B: Cis-acting elements of PmARF17 promoter "

Fig. 4

Expression analysis of PmARF17in flower development of Prunus mume A: PmARF17 expression in flower buds and leaf buds of Daqiandi from September to January; B: PmARF17 expression in flower buds and leaf buds of Longyan from September to January; C: PmARF17 expression in flower buds of different pistil morphologies of Daqiandi; D: PmARF17 expression in different mature flower organs of Daqiandi "

Fig. 5

Correlation analysis between hormones and PmARF17 expression of flower buds and leaf buds at different periods of Daqiandi and Longyan A: IAA, GA3, ABA, ZT contents and the PmARF17 expression of flower buds and leaf buds at different periods of the aborted Daqiandi; B: IAA, GA3, ABA, ZT contents and the PmARF17 expression of flower buds and leaf buds at different periods of well-developed Longyan. * indicate significant difference ( P<0.05), and ** indicate extremely significant difference (P<0.01). The same as below "

Fig. 6

Analysis of endogenous hormone contents and PmARF17 expression in flower buds of different pistil morphologies and different mature flower organs of ‘Daqiandi’ A: IAA, GA3, ABA, ZT contents and the PmARF17 expression in flower buds of different pistil morphologies of Daqiandi; B: IAA, GA3, ABA, ZT contents and the PmARF17 expression in different mature flower organs of Daqiandi "

Fig. 7

The structure of PmARF17 promoter-GUS vector and transient expression experiment of PmARF17promoter A: The structure of pBI121-PmARF17 promoter-GUS vector; B: GUS staining results of Arabidopsis thaliana seedlings infected by pBI121-PmARF17 promoter-GUS vector; C: Negative control GUS staining results (without bacterial infection); D: Positive control GUS staining results (infected by bacterial fluid of pBI121 alone); E: Enlarged view of the root in B; F, G: GUS staining results of pBI121-PmARF17 promoter-GUS in petals; H: GUS staining results of pBI121-PmARF17 promoter-GUS in stamens; I: High magnification image of blue area separated from H; J: GUS staining results of floral organ in the negative control, and its stamens have the same components as those in I; K, L: GUS staining results of different roots, flower organs and leaves in the positive control "

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