【作者】 苏群；

【导师】 唐开学；

【作者基本信息】 云南大学 ， 植物学， 2016， 硕士

【摘要】 月季属于蔷薇科(Rosaceae)蔷薇属(Rosa L.)植物,是世界四大切花之一,具有重要的经济价值。以月月粉(Rosa chinensis’Old Blush’)为代表的中国古老月季品种因具有连续开花的习性而深受人们的喜爱,也为现代月季育种贡献了优良性状。花器官是衡量月季观赏价值的重要指标之一,研究表明MicroRNA172(miR172)与其靶基因AP2在植物花器官发育过程中发挥重要作用,AP2等基因功能沉默会导致花器官的发育异常。本研究以‘月月粉’为研究材料,拟建立基于TaqMan探针技术的月季microRNA表达谱分析技术体系,并初步分析miR172在‘月月粉’部分花器官中的表达水平。具体研究结果如下：(1)研究通过对microRNA数据库(http://www. mirbase. org/)同源性搜索,获得了月季miR172家族(miR172a和miR172c)成熟体基因序列,并和蔷薇科苹果、湖北海棠、碧桃等物种进行序列比对分析,分析结果显示月季的miR172基因和其他蔷薇科miR172基因有着较高的同源性。(2)研究以‘月月粉’花芽分化期茎尖转录组序列的41617个已经注释的Unigene为参考序列,利用植物靶基因预测软件psRobot及TargetFinder对miR172进行靶基因及靶基因前体预测,共获得14个候选靶基因及13个前体基因序列。对候选靶基因进行功能注释,以期为下一步研究miRl72及其靶基因的作用模式及功能研究奠定一定理论基础。(3)研究以月季5S基因为参照基因,基于TaqMan探针技术完成了miR172家族在24小时内,6个时间点‘月月粉’花药和花柱(包含柱头,下同)的表达模式分析。研究结果显示：miR172a在花柱中呈现双峰的表达模式,分别出现在12：00和18：00,其中尤以中午12：00表达量最高。此外miR172a在花柱中的日表达量变化呈现出较为规律性的波动；miR172c在6个取样时间点花柱中没检测到显著的表达水平,仅在上午8：00时检测到0.68的表达量。(4)研究以月季5S基因为参照基因,采用TaqMan探针技术完成了miR172家族在‘月月粉’不同发育阶段及同一阶段不同时间点的花瓣表达量分析。结果显示miR172在不同发育阶段的花瓣中相对表达量不相同,且随着花瓣生长和开放程度的增加,miR172表达量逐渐升高。在不同发育阶段花瓣中,miRl72家族日最高表达量均出现在中午14：30,miR172家族在不同阶段花瓣中的相对表达量呈现出一定的规律性波动。可能由于天气的原因(阴天,无光照)第二次生物学重复当天的相对表达量较低,需要在将来的研究中进一步给予确认。更多还原

【Abstract】 Chinese rose belongs to the family Rosaceae and genus Rosa, is one of the four big cut flowers in the world and has important economic value.Rosa chinensis’Old Blush’is a famous ancient diploid variety of China rose.Its good qualities deeply loved, and the excellent characters are contributed to Chinese rose breeding.Flower type is one of the important indicators to measure Chinese rose ornamental value. Studies have shown that MicroRNA172 (miR172) and their target genes AP2 involved in plant reproductive phase shift,and its function silence of target genes also can lead to abnormal flower organs. Rosa chinensis’Old Blush’was studied as the research material, the expression patterns of microRNA172 (miR172) family were investigated preliminarilytion along the developmental process of flower’anther, style and petals in the Chinese rose.The main results were as follows:(1)The mature miR172 gene family sequences (miR172a and miR172c) of Rosa were obtained by homology searching in mirna database (http://www.mirbase.org/), and compared with Prunus persica,Malus domestica and Malus hupehensis, The results showed that the Chinese rose miR172 gene and other Rosaceae miR172 genes were high homology.(2) 41617 Unigenes of stem tip transcriptome sequences from Rosa chinensis’Old Blush’flower bud differentiation period were studied as reference sequences, miR172 target genes were predicted by using plant target gene prediction software psRobot.14 higher degree of feasible target genes and 13 precursor sequence were gotten, and made as foundation for the action mode study between miR1 72 and target genes and functional research.(3) 5 s gene was made as a reference gene, basing on TaqMan probe technology miR1 72 expression modal analyses of 16 hours,6 time-points in Rosa chinensis’Old Blush’anther and stylel were completed, the results showed that:miR172a expression had two peaks in one day, at 12:00 and 18:00 respectively, especially at 12:00 with the highest relative expression.In addition,MiR172a express are relatively regular fluctuations in the style of the day.but miR172c hardly expressed in 16 hours one day, though at 8:00 detected maximum expression instyle, just 0.68, the total expression quantities were very low.(4) 5 s gene was made as a reference gene, basing on TaqMan probe technology, the amount of miR172 expression in petals’different development stages of Rosa chinensis’Old Blush’were completed.Research results showed that the amounts of miRl 72 expression in the different development stages were not identical, along with the petals growthing and openness increasing, miR172 expression gradually rised.In different development phase, the expression of miR172a and miR172c showed time rhythm phenomenon, the the highest relative expression were happened every day at noon.May be because of the weather (cloudy, no light), the second time biology repeat showed low relative expression in one day, so further study is needed in the future to confirm the reason.更多还原