甘蓝ROH1与EXO70A1的表达与相互作用

Expression and Interaction Between ROH1 and EXO70A1 in Reproductive Development

【目的】以自交不亲和材料甘蓝为研究对象,分析ROH1的组织表达、ROH1与EXO70A1的相互作用及其是否参与甘蓝生殖发育。【方法】从甘蓝花蕾中克隆出ROH1和EXO70A1,应用半定量RT-PCR检测ROH1的表达特性;应用实时荧光定量PCR进一步分析甘蓝授粉后1 h内ROH1和EXO70A1的表达量和二者的相关性;分别构建以p GBKT7为载体的EXO70A1重组"诱饵"质粒和以p GADT7为载体ROH1的重组猎物质粒,并将重组质粒转化酵母菌株,利用缺陷型培养基进行蛋白质相互作用检测,确定ROH1和EXO70A1是否存在相互作用。【结果】在甘蓝中ROH1为单外显子基因,编码含398个氨基酸残基的蛋白质,与拟南芥ROH1对比发现,甘蓝ROH1序列内存在一个连续16个氨基酸残基的缺失;它在甘蓝的雄蕊(花药)、花柱、幼茎、幼根及叶片中均有表达但表达量差异明显,其中,在幼叶、花柱和雄蕊(花药)中的表达量较高,在幼根和幼茎中的表达量较低;甘蓝授粉后柱头中ROH1的表达量在1 h内呈现出"上升-下降-上升"趋势,其中,以授粉后30 min的表达量最低,授粉后1 h的表达量达到最高值;而EXO70A1在授粉后1 h内的表达呈现出"下降-上升"的趋势,并以授粉15 min时表达量最低,授粉后1 h时的表达量最高;二者表达的动态变化说明ROH1和EXO70A1参与了甘蓝的生殖发育;ROH1与EXO70A1的表达量趋势线呈现出负相关性,并在30 min时出现了重叠区域,预示ROH1与EXO70A1之间可能存在相互作用;成功构建p GADT7-ROH1和p GBKT7-EXO70A1酵母表达载体,通过酵母双杂交试验,发现载体p GBKT7-EXO70A1无自激活能力,融合菌株同时激活4个报告基因(AUR1-C、MEL1、HIS3和ADE2)的表达,表明花药中ROH1和花柱中的EXO70A1之间存在较强的相互作用。【结论】甘蓝的ROH1和EXO70A1之间存在较强的相互作用并呈现出负相关性,ROH1可能通过调节EXO70A1在柱头的分泌影响生殖发育。

[Objective] The objectives of this study were to clone ROHland EXOTOA1 from the Brassica oleracea bloom, and establish whether EXO70A1 and ROH1 are involved in the reproductive development ofBrassica oleracea. [Method] The coding sequences of ROH1 were cloned by PCR from Brassica oleracea. In addition, the coding sequences of EX070.41 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 EXO7OA1 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 Exo7OA1 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 ofROH1 gene increased between 0-15 min, decreased from 15 min, and peaked at 1 hour post-pollination The expression of EXO70A 1 gene tracked ROH expression pattern. The changes in expression of EXO7OA1 and ROH1 implied that they were involved in reproductive development. The expression of ROH1 and EXO7OA1 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.