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Cloning and Expression Analysis of BoSPI Induced by Self-pollination in Brassica oleracea L. var. capitata
Acta Agronomica Sinica
2018, 44 (02):
177-184.
DOI: 10.3724/SP.J.1006.2018.00177
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. ![]()
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.
Extracts from the Article
图3 BoSPI基因序列及其推导的氨基酸序列(A)、Ca2+结合核心结构分析(B)和BoSPI蛋白三维结构预测(C)
A图中下画线指示4个EF-hand结构域, 红色字体表示12个氨基酸组成的Ca2+结合的核心区域; B图中下排数字表示对这12个氨基酸残基的编号, 数字0表示该核心区域上游第一个氨基酸残基。
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