【作者】 高峰；

【导师】 朱英国； 李绍清；

【作者基本信息】 武汉大学 ， 遗传学， 2015， 博士

【摘要】 本研究通过对miR396b和GRF6元件之间的调控关系及该调控元件调控的下游基因和代谢通路进行研究,初步揭示了miR396b/GRF6、激素和花序形态建成之间的关系,具体结果如下：1)通过对红莲型杂交稻组合的幼穗中miRNA表达量进行系统分析,发现miR396为调控水稻枝梗分化的关键miRNA。过表达miR396b造成水稻植株矮小,穗长变短,二次枝梗消失,颖花成簇聚集,而miR396干涉的植株则表现出相反的表型：株型紧凑,穗长增加,二次枝梗数目显著增加56.4±16.6%,(P<0.001),单株产量显著增加41.8±11.1%,(P<0.001)。2)MiR396能够调控其靶标基因GRF家族中OsGRF6的表达水平,转录因子OsGRF6的高水平表达能够使水稻的二次枝梗数目显著增加59.3±12.9%(P<0.001),每穗粒数显著增加54.3±9.1%(P<0.001),明显增加水稻的单株产量47.6±15.7%(P<0.001),而对OsGRF6的干涉造成水稻二次枝梗的减少甚至消失,进而使每穗粒数显著减少,降低水稻产量。3)在水稻OsGRF6过表达的转基因株系中,其单株产量显著增加,这种增产现象同样在我们的大田试验中得到验证,在武汉进行的大田试验中,MIM396和OEGRF6的转基因株系分别比野生型增产16.8±0.2%和18.1±0.2%,而在海南进行的大田试验中,MIM396和OEGRF6过表达的转基因株系分别比野生型增产15.4±0.2%和16.0±0.2%,通过两地的大田试验说明过表达OsGRF6能够使水稻的单位产量显著增加。4)转录因子OsGRF6能够调控一系列的下游基因及其涉及到的代谢通路,有生长素生物合成和信号传递的相关基因OsYUCCA1、OsARF2、OsARF7和OsARF11,还有两个调控水稻幼穗发育的关键基因OsTAWAWA1和OsMADS34同样受到OsGRF6的调控。在MIM396和OEGRF6的转基因材料中,这些受到OsGRF6调控的下游基因都出现表达量明显提高,使得枝梗发育时期幼穗中生长素的含量显著提高,而生长素含量的提高增加了枝梗原基的分化,进而分化出更多的分支原基和颖花,提高水稻的产量。而OsTAWAWA1和OsMADS34这两个水稻幼穗发育关键基因表达量的提高,同样能够增加水稻的二次枝梗数目和每穗颖花数,增加水稻产量。通过对红莲型杂交稻幼穗中miRNA的分析,我们发现miR396作为调控水稻枝梗发育关键的miRNA, miR396调控靶标基因OsGRF6的表达,并且miR396b/OsGRF6这一调控元件对下游生长素合成和信号应答基因及幼穗发育关键基因OsTAWAWA1和OsMADS34进行调控。这一调控通路揭示了miRNA、植物激素和幼穗的枝梗分化这三者之间的联系。通过生物工程技术,对这三者之间进行动态的平衡,并在分子育种方面,为我们提高水稻产量打下重要的基础。更多还原

【Abstract】 The yield of hybrid F1 increased about 20% than their parents in Honglian-type hybrid rice. To discover the mechanism of increased yield of Honglian-type hybrid rice is very important for improving the innovation of moleculer breeding theory and practice production.Here, we discovered the relationship between miR396b and OsGRF6, along with the downstream genes and metabolic pathway that regulated by this module. Revealed that the connection between miR396b/GRF6 module, hormone and inflorescence arcchitecture. The results as follow in detail:1) After investigated miRNAs expression level in Honglian-type hybrid rice, we found that miR396 regulated branch differentiation as a key player. Overexpression of miR396b transgenic plants displayed dwarf stature, abnormal inflorescence without the secondary branches and pedicels, and the spikelets were clustered together and sparsely scattered across the primary branches, on the contrary, MIM396 transgenic plants showed plant morphology compact, and a significant increase in yield (41.8±11.1%, P <0.001) compared to that of the wild type, which might have been due to the substantial increase in the number of secondary branches (56.4±16.6%, P<0.001).2) MiR396 regulated the expression level of target gene GRF6, as a transcript factor, high level expressed GRF6 increased the yield by 47.6±15.7%(P<0.001) compared to that of the WT, In OEGRF6-1 transgenic plants, the number of secondary branches and spikelets increased by 59.3±12.9%(P<0.001), and 54.3±9.1%(P< 0.001), respectively. In GRF6 interferential transgenic plants, the secondary branches were decreased, even disappeared, and then decreased the number of grain per panicle and yield per plant.3) The yield were increased in overexpression transgenic plant lines. We verified these results in field tests. The yield of MIM396-9 and OEGRF6-1 transgenic lines increased 16.8±0.2% and 18.1±0.2% than WT in Wuhan, and 15.4±0.2% and 16.0 ±0.2% in Hainan. These results confirmed that high expression level of GRF6 could increase yield significantly.4) Transcript factor OsGRF6, regulated the expression of downstream genes that the metabolic pathway relevant. Which coordinately regulated transcription of downstream OsMADS34 and OsTAWAWA1 involving in regulated the secondary branch development, and OsYUCCAl, OsARF2, OsARF7 and OsARF11 related to AUX/IAA biosynthesis and downstream signaling. The expression level of these genes above were increased, and then enhance the content of AUX/IAA in MIM396 and OEGRF6 transgenic plants young inflorescence. The increased branch primordia differentiated more lateral branch primordia and spikelet primordia, because the enhanced AUX/IAA, and then increased rice yield. OsTAWAWA1 and OsMADS34, as the key genes to regulate young inflorescence development, improved rice yield by increased the number of secondary branch and spikelet per panicle.After the analysis of miRNAs in Honglian-type hybrid rice young inflorescence. Our study suggest that OsmiR396/OsGRF6 module act as a key player in regulated transcription of downstream genes OsMADS34 and OsTAWAWAl involving in the secondary branch development, and OsYUCCA1, OsARF2, OsARF7 and OsARF11 related to AUX/IAA biosynthesis and downstream signaling pathway. To discover the relationship between miRNA, hormone and branch differentiation, offered a novel way to engineering high yield rice in moleculer breeding.更多还原