Scientia Agricultura Sinica ›› 2018, Vol. 51 ›› Issue (22): 4328-4338.doi: 10.3864/j.issn.0578-1752.2018.22.011

• HORTICULTURE • Previous Articles     Next Articles

Cloning and Expression Analysis of BoSPx in Brassica oleracea

WANG YuKui1(),BAI XiaoJing1,LIAN XiaoPing2,ZHANG HeCui1,LUO ShaoLan1,PU Min1,ZUO TongHong1,LIU QianYing1,ZHU LiQuan1()   

  1. 1 College of Agronomy and Biotechnology, Southwest University, Chongqing 400715
    2 College of Horticulture and Landscape Architecture, Southwest University, Chongqing 400715
  • Received:2018-04-21 Accepted:2018-07-21 Online:2018-11-16 Published:2018-11-16

Abstract:

【Objective】Self-incompatibility (SI) is a genetic barrier to inhibit self-pollination and promote hybridization in flowering plants. Here, cloning of a novel gene co-responsive to SI and calcium production during pollination, and spatio-temporal specific expression analysis of the novel gene BoSPx under self-pollination conditions, screening its interaction proteins were conducted to explore. The responding mechanism of BoSPx to self-pollination stimulated stigma in order to provide some further insights into SI process in Brassica oleracea.var.Capitata.【Method】BoSPx was cloned by using transcriptome sequencing, self-pollination and cross-pollination differential screening, and PCR cloning. Amino acid sequence alignment and conserved domain analysis were performed by DNAMAN and Smart software. Expasy online software was used to predict BoSPx protein molecular weight, isoelectric point, secondary structure and transmembrane domain. Phylogenetic tree that constructed by the neighboring method in MEGA6.0 software was used to speculate on the function of BoSPx protein after self-pollination. RT-PCR and qRT-PCR were used to detect BoSPx tissue-specific expression and the relative expression of BoSPx after self-pollination and cross-pollination. The BoSPx-GFP expression vector was constructed and the subcellular localization of BoSPx was observed under confocal microscopy; The interaction proteins were searched by using yeast two-hybrid system. 【Result】A novel gene, which contains a single exon without any introns, named BoSPx was cloned. The open reading frame of BoSPx is 396 bp, encodes a protein with 131 amino acid residues. BoSPx is a hydrophilic protein, no signal peptide and transmembrane, and the theoretical isoelectric point is 4.54. Conserved domains analysis found BoSPx contains three conserved EF-hand motifs (48-60, 64-80, and 81-96). About 500 bp up-stream of BoSPx translation start code contains an auxin response element. RT-PCR analysis found that BoSPx was expressed highest in stigma, BoSPx was also expressed in sepals, leaves, anthers and petals in flowering stage. The BoSPx expression levels in both self-pollinated and cross-pollinated stigma showed “up-down-up” expression pattern. Moreover, The expression of BoSPx in the stigma of self-pollination and cross-pollination increased at first and then decreased down to the highest expression level in stigma in flowering stage. The expression of BoSPx increased rapidly after self-pollination at 15 min and then decreased sharply so that result in SI progress. The decreased value of BoSPx was 1-2 days before flowering. Subcellular location analysis found that BoSPx expression in both the nucleus and cytoplasm. Yeast two-hybrid system did not detect interaction between BoSPx and SRK and ARC1, but BoSPx interacted with auxin family proteins BoSAUR71 and BoPID. 【Conclusion】BoSPx is highly expressed by self-pollination, which may be an auxin-regulated calcium-binding protein, which has a common response to SI and calcium. The protein has multi-tissue expression and nuclear and cytoplasmic properties, indicating that BoSPx may be involved in unknown signaling pathways other than the SRK-ARC1-ExO70A1 pathway.

Key words: Brassica oleracea, BoSPx, yeast two-hybrid, auxin, self-pollination, self-incompatibility

Fig. 1

Transcriptome data were analyzed for expression patterns of BoSPx after self-pollination and cross- pollination SP: Self-pollination; CP: Cross-pollination. The same as below"

Fig. 2

Amplification of BoSPx gene from cDNA and gDNA of the stigma of Brassica oleracea"

Fig. 3

Structural characteristics of BoSPx protein in Brassica oleracea A:BoSPx gene sequence and deduced amino acid sequence; B: Analysis of EF-hand domain features of BoSPx using a consensus sequence analysis with MARSDEN et al[22]; C: BoSPx protein three-dimensional structure prediction"

Fig. 4

Phylogenetic tree of BoSPx and other species BoSPx amino acid sequence"

Fig. 5

Multiple alignment of deduced amino acid sequence of BoSPx with homologous proteins of other species BnSPx: XP_013685948; RsSPx: XP_018468047; BrSPx: XP_009127085; The underlined representation of the EF-Hand domain also indicates that the protein is highly conserved in this region"

Table 1

Cis-elements in the upstream regulation region of BoSPx gene"

相关功能预测
Associated putative function
启动子顺式作用元件
Cis-elements in the promoter region
脱落酸响应元件Abscisic acid responsiveness ABRE
光响应元件Light responsive element ATCT-motif, Box4, G-Box, ACE, CATT-motif, GAG-motif
Gap-box, TCCC-motif, I-box, MNF1, TCT- motif, as-2-box
顺式作用调节元件Cis-acting regulatory element ARE
启动子和增强子区域Promoter and enhancer regions CAAT-box
热应激反应Heat stress responsiveness HSE
低温响应元件Low-temperature responsiveness LTR
干旱诱导反应元件Drought induced response element MBS
调控胚乳表达元件Regulatory element for endosperm expression GCN4_motif, Skn-1_motif
水杨酸响应元件Salicylic acid responsiveness TCA-element
茉莉酸响应元件MeJA-responsiveness CGTCA-motif, TGACG-motif
生长素响应元件Auxin-responsive element TGA-element

Fig. 6

RT-PCR analysis of BoSPx gene in different tissues of Brassica oleracea"

Fig. 7

Expression analysis of BoSPx in response to self and cross pollination"

Fig. 8

Subcellular localization of BoSPx protein in Arabidopsis protoplasts"

Fig. 9

The Co-Transformed plasmid in yeast grown on SD/-Leu/-Trp plate"

Fig. 10

Test protein-protein interactions on SD/-Ade/-His/- Leu/-Trp plate"

Table 2

Interaction analysis of plasmid co-transformation yeast cell"

编号
No.
酵母菌种(质粒)
Mating strain (plasmid)
培养基
Yeast medium
菌斑
Colony
颜色
Color
1 Y2HGold(pGADT7-T×pGBKT7-53) SD/-Leu/-Trp 是Yes 白色White
2 Y2HGold(pGADT7-T×pGBKT7-Lam) SD/-Leu/-Trp 是Yes 红色Red
3 Y2HGold (pGADT7-SRK×pGBKT7-BoSPx) SD/-Leu/-Trp 是Yes 红色Red
4 Y2HGold (pGADT7-ARC1×pGBKT7-BoSPx) SD/-Leu/-Trp 是Yes 红色Red
5 Y2HGold (pGADT7-SAUR71×pGBKT7-BoSPx) SD/-Leu/-Trp 是Yes 白色White
6 Y2HGold (pGADT7-BoPID×pGBKT7-BoSPx) SD/-Leu/-Trp 是Yes 白色White
7 Y2HGold(pGADT7-T×pGBKT7-53) SD/-Ade/-His/-Leu/-Trp 是Yes 白色White
8 Y2HGold(pGADT7-T×pGBKT7-Lam) SD/-Ade/-His/-Leu/-Trp 是Yes 红色Red
9 Y2HGold (pGADT7-SRK×pGBKT7-BoSPx) SD/-Ade/-His/-Leu/-Trp 无No 无No
10 Y2HGold (pGADT7-ARC1×pGBKT7-BoSPx) SD/-Ade/-His/-Leu/-Trp 无No 无No
11 Y2HGold (pGADT7-SAUR71×pGBKT7-BoSPx) SD/-Ade/-His/-Leu/-Trp 是Yes 白色White
12 Y2HGold (pGADT7-BoPID×pGBKT7-BoSPx) SD/-Ade/-His/-Leu/-Trp 是Yes 白色White
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