自花授粉诱导的甘蓝功能基因BoSPI的克隆与表达分析

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

自交不亲和性是甘蓝在长期进化过程中形成的防止自交衰败、促进杂交优势的一种复杂而完善的遗传机制。克隆自交不亲和性相关基因对甘蓝自交不亲和性的深入研究和利用有重要意义。本研究通过挖掘0~60 min自花和异花授粉的甘蓝柱头转录组数据,筛选到一个受自花授粉诱导上调表达的基因,命名为BoSPI。BoSPI开放阅读框534bp,编码177个氨基酸,理论等电点为4.21,不包含信号肽和跨膜区,含有4个保守的EF-hand结构域。BoSPI基因起始密码子上游2000 bp的核苷酸序列中含有真菌诱导响应、代谢调节以及器官形成等应答元件。BoSPI基因在大肠杆菌中可诱导表达为17 k D的蛋白。BoSPI在柱头中表达量最高,在花瓣、萼片、叶片、雄蕊表达量较低。BoSPI蛋白被定位在细胞膜和细胞质。自花授粉30 min后对BoSPI基因的诱导表达显著增强。表明BoSPI参与了柱头响应自花花粉刺激的分子过程,可能是实现甘蓝自交不亲和性相关的某种新功能基因。

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.