甘蓝ROH1与EXO70A1的表达与相互作用

doi:10.3864/j.issn.0578-1752.2016.04.016

Abstract

Abstract: 【Objective】The objectives of this study were to clone ROH1 and EXO70A1 from the Brassica oleracea bloom, and establish whether EXO70A1 and ROH1 are involved in the reproductive development of Brassica oleracea. 【Method】The coding sequences of ROH1 were cloned by PCR from Brassica oleracea. In addition, the coding sequences of EXO70A1 was cloned from Brassica oleracea mRNA. RT-PCR was used to analyze expression characteristics of ROH1, while Real Time PCR was used to analyze expression of ROH1 and EXO70A1 in 1 hour post-pollination. A Yeast Two-hybrid System was used to determine the interaction between ROH1 and EXO70A1.The encoding sequences of ROH1 was subcloned into vector pGADT7 and Exo70A1 was subcloned into vector pGBKT7. Then the interaction between ROH1 and EXO70A1 was tested in selection medium by yeast two-hybrid System.【Result】We demonstrated that the ROH1 gene is a single-exon gene encoding a 398-amino-acid protein in Brassica oleracea. With the ROH1 in Arabidopsis, the sequence losses 16 amino acid residues in Brassica oleracea, In addition, ROH1 expressed in stems, styles, anther, young roots and leaves. Notably, expression levels varied in different tissues, with strong expression in styles, anthers and leaves, but with weak expression in stems and roots. After pollination, the expression of ROH1 gene increased between 0-15 min, decreased from 15 min, and peaked at 1 hour post-pollination The expression of EXO70A1 gene tracked ROH expression pattern. The changes in expression of EXO70A1 and ROH1 implied that they were involved in reproductive development. The expression of ROH1 and EXO70A1 appeared in overlapping region at 30 min. This overlap suggested that a potential interaction between ROH1 and EXO70A1 in Brassica olerace. The recombinant expression vectors did not exhibit autoactivation and toxicity. Yeast Two-hybrid system experiments established that the recombinant expression vectors could grow well on nutritional media without transcription activation of the reporter genes AUR1-C, MEL1, HIS3 and ADE2, and it showed an interaction between ROH1 and EXO70A1 in Brassica oleracea.【Conclusion】The strong interaction between ROH1 was detected during reproductive development in Brassica oleracea. It is speculated that ROH1 and EXO70A1 affects the reproductive development by regulating the secretion of the stigma EXO70A1, putting forward a novel insight into the reproductive development process in Brassica oleracea system.

Key words: Brassica oleracea, ROH1, EXO70A1, yeast two-hybrid system

ZHANG He-cui, LIU Jing, LIAN Xiao-ping, ZENG Jing, YANG Kun, ZHANG Xue-jie, YANG Dan, SHI Song-mei, GAO Qi-guo, ZHU Li-quan. Expression and Interaction Between ROH1 and EXO70A1 in Reproductive Development[J].Scientia Agricultura Sinica, 2016, 49(4): 775-783.

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