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【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 objective of this study was to investigate the expression of ROH1, the interaction between ROH1 and EXO70A1 and whether ROH1 participates in cabbage reproductive development. 【Method】 ROH1 and EXO70A1 were cloned from cabbage flower buds, and the expression of ROH1 was detected by semi-quantitative RT-PCR. The expression of ROH1 and EXO70A1 within 1 h after pollination were analyzed by real-time fluorescence quantitative PCR and their correlation ; Construction of recombined “Bait” plasmid with EXT70A1 vector and pGADT7 as the carrier of ROH1 recombinant plasmid pGBKT7, respectively, and the recombinant plasmid was transformed yeast strains, the detection of protein-protein interactions using defective medium ROH1 Interaction with EXO70A1. 【Result】 ROH1 was a single exon gene in Brassica oleracea and encoded a protein with 398 amino acid residues. Compared with ROH1 in Arabidopsis thaliana, ROH1 in Brassica oleracea showed a deletion of one consecutive 16 amino acid residues in ROH1. (Anthers), styles, young stems, young roots and leaves, but their expression levels were significantly different. Among them, the expression levels in young leaves, style and stamens (anthers) were higher than those in young roots and young stems The expression level of ROH1 in the stigma of pollination showed a trend of “rising-falling-rising” in 1 h after pollination, in which the expression level of ROH1 was the lowest at 30 min after pollination and at 1 h after pollination The highest expression level of EXO70A1 was observed within 1 h after pollination, and the expression of EXO70A1 tended to decrease by 15 min after pollination and highest at 1 h after pollination The dynamic changes in expression of ROH1 and EXO70A1 were involved in the reproductive development of cabbage. The trend lines of ROH1 and EXO70A1 expression showed a negative correlation, and the overlapping region appeared at 30 min, indicating that there may be interaction between ROH1 and EXO70A1. The pGADT7-ROH1 and pGBKT7-EXO70A1 yeast expression vectors were successfully constructed, After yeast two-hybrid assay, the vector p GBKT7-EXO70A1 was found to be non-self-activating, and the expression of four reporter genes (AUR1-C, MEL1, HIS3 and ADE2) was simultaneously activated by the fusion strain, indicating that between the anther ROH1 and EXO70A1 in the style There is a strong interaction. 【Conclusion】 There is a strong negative correlation between ROH1 and EXO70A1 in cabbage, and ROH1 may affect reproductive development by regulating the secretion of EXO70A1 at the stigma.