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Acta Agronomica Sinica ›› 2018, Vol. 44 ›› Issue (02): 177-184.doi: 10.3724/SP.J.1006.2018.00177

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Cloning and Expression Analysis of BoSPI Induced by Self-pollination in Brassica oleracea L. var. capitata

Min PU1, Shao-Lan LUO1, Xiao-Ping LIAN2, He-Cui ZHANG1, Xiao-Jing BAI1, Yu-Kui WANG1, Tong-Hong ZUO1, Qi-Guo GAO2, Xue-Song REN2, Li-Quan ZHU1,*()   

  1. 1 College of Agronomy and Biotechnology, Southwest University, Chongqing 400715, China
    2 Key Laboratory in Olericulture of Chongqing, Chongqing 400715, China
  • Received:2017-05-04 Accepted:2017-09-10 Online:2018-02-12 Published:2017-10-27
  • Contact: Li-Quan ZHU E-mail:zhuliquan@swu.edu.cn
  • Supported by:
    This study was supported by the National Natural Science Foundation of China (31572127) and the Research Project of Graduate Students in Chongqing (CYS16076).

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Abstract:

Self incompatibility is a complex and comprehensive genetic mechanism formed in the long-term evolution, which prevents inbreeding and promotes heterosis. Mining functional genes involving in self incompatibility has an important significance for the study of self incompatibility in Brassica oleracea L. var. capitata. In this study, we identified a gene named BoSPI which expression was up-regulated and induced by self-pollination based on the stigma transcriptome data in 0-60 min self-pollination and cross-pollination. BoSPI contains an open reading frame (ORF) with the length of 534 bp, encoding a protein of 177 amino acid residues without introns, which contains four conserved EF-hand domains without signal peptide and transmembrane domain, the theory isoelectric point of BoSPI is 4.21. In addition, diverse cis-acting promoter elements involved in fungal elicitor response, metabolic regulation and organ formation were discovered in the upstream 2000 bp of initial codon of BoSPI. BoSPI could be expressed as a 17 kD protein in E. coli BL21 (DE3). The expression level of BoSPI was the highest in stigmas and lower in petals, sepals, leaves and stamens of cabbage after self-pollination. Subcellular localization analysis revealed that BoSPI encoded a protein localized in the cell membrane and cytoplasm. The expression of BoSPI gene was significantly induced by self pollination after 30 minutes. These results suggest that BoSPI is involved in the molecular processes of the stigma response to self-pollen stimulation, which may be a new functional gene related to the self incompatibility of Brassica oleracea L. var. capitata.

Key words: self-pollination, Brassica oleracea L. var. capitata, gene (BoSPI), transcriptome technology

Table 1

Primers used in gene cloning and RT-PCR"

引物
Primer		 引物序列
Primer sequence (5′-3′)		 退火温度
Tm (ºC)		 引物用途
Primer description.
GST F: GATCTGGTTCCGCGTGGATCCATGGCAAGTGGTAACCCTGTAAC 60 基因的原核表达
Prokaryotic expression
R: GATGCGGCCGCTCGAGTCGACCTAGGCCGTCGAACCACCG
qRP F: GATCAGGACAAGAACGGGCTC 60 qRT-PCR
R: GTTGTTGCTGTTAGCGAGGGAG
Actin3 F: GGCTGATGGTGAAGATATTCA 58 内参引物
Internal reference
R: CAAGCACAATACCAGTAGTAC
GFP F: AAGTCCGGAGCTAGCTCTAGAATGGCAAGTGGTAACCCTGTAACC 63 基因亚细胞定位
Subcellular localization of gene
R: GCCCTTGCTCACCATGGATCCGGCCGTCGAACCACCGTTC

Fig. 1

Expression pattern of BoSPI in stigma of cabbage in response to self and cross pollination by transcriptome technology"

Fig. 2

Amplification of BoSPI gene from cDNA and gDNA of the stigma of cabbage"

Fig. 3

BoSPI cDNA sequence and deduced amino acid sequence (A), analysis of the core area of calcium ion binding (B) and predicted three-dimensional structure (C) for BoSPI protein The underlined sequence means four EF-hand domains and the read marker means the core area of calcium ion binding in Fig. A; the numbers indicate the numbers of the 12 amino acid residues of the core area of calcium ion binding and the digital 0 represents the first amino acid residue upstream of the core region in Fig. B."

Fig. 4

Phylogenetic relationship of nucleotide sequence between BoSPI and its homologous genes The red boxs indicate the nucleotide site that is different from CML27 but identical with CML26."

Table 3

cis-elements in the upstream regulation region of BoSPI gene"

相关功能预测
Associated putative function		 启动子顺式作用原件
cis-elements in the promoter region
Light response ACE, ATCT-motif, Box 4, Box I, G-Box, GAG-motif, MRE
Salicylic acid response TCA-element
MeJA response CGTCA-motif, GARE-motif
Endosperm expression Skn-1_motif
Fungal elicitor response Box-W1
Gibberellin response GARE-motif
DNA binding protein (ATBP-1) AT-rich element
Anaerobic induction ARE
Abscisic acid response ABRE
Stress responsiveness HSE, LTR, MBS, TC-rich repeats

Fig. 5

Prokaryotic expression protein analysis of BoSPI protein M: marker; 1: GST protein; 2: BoSPI-GST fusion protein."

Fig. 6

Subcellular localization of BoSPI-GFP protein in tobacco cell Green indicates the green fluorescence of GFP protein under confocal laser scanning microscope."

Fig. 7

Expression analysis of BoSPI in different organs of Brassica oleracea L. var. capitata"

Fig. 8

Expression level of BoSPI in response to self and cross pollination SI: self-pollination; CP: cross-pollination."

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