水稻OsFBHs的功能及其分子机理研究

发布时间：2018-05-25 02:38

本文选题：水稻 + 拟南芥　；参考：《沈阳农业大学》2017年博士论文

【摘要】：开花期在水稻(Oryza sativa)发育和产量形成过程中起到非常重要的作用。水稻开花期是内源因素和外界的环境条件相互作用、多基因参与的、复杂的生命过程。虽然,目前已经初步建立了 Heading date 1和Early heading date 1为关键调节因子的促进或抑制水稻开花的分子调控网络。但是,水稻开花期调控的分子机制还远不清楚。本项目借助生物信息学方法,利用拟南芥(Arabidopsis thaniala)中开花时间相关基因Flowering bHLHs(FBHs)基因家族的序列,筛选获得了水稻中的同源基因OsFBHs;利用CRISPR-Cas9基因编辑技术、DNA重组和水稻转基因技术,创制了OFBVHs基因超表达、下调表达或基因定点敲除的水稻或拟南芥转基因材料;经过对转基因株系的农艺性状观察,筛选获得了开花期等农艺性状发生变化的OsFBH1和OsFBH3基因的转基因株系。利用这些开花期改变的转基因株系为试材,采用荧光定量RT-PCR,酵母单(双)杂交,染色质免疫共沉淀(Chromatin Immunoprecipitation,ChIP)等技术,本项目深入研究了OsFBH 的表达模式、亚细胞定位、调控开花期的靶基因;揭示了 OsFBHs调控水稻开花期的分子机制。为不同生育期的水稻品种改良,提高水稻的适应性、产量和品质提供理论依据。本项目的主要研究结果如下:1.水稻OsFBHs与拟南芥FBHs基因进化上保守,而且亲缘关系较近。拟南芥中FBHs基因家族有4个成员(包括:FBH1-4-),编码包含有HLH保守结构域的蛋白质。水稻中有4个FBHs的同源基因(OsFBH1-4)与拟南芥中的FBHs亲源关系较近。其中OsFBH1和OsFBH2与拟南芥的FBH1和FBH2的亲缘关系较近,OsFBH3和OsFBH4与拟南芥中的FBH3和FBH4的亲缘关系较近。2.OsFBH1的表达模式和亚细胞定位分析。荧光定量RT-PCR与GUS染色的结果表明,OsFBH1在水稻的叶片和叶鞘中表达水平较高,在根部和胚芽鞘组织中也有表达。OsFBH1的表达受生物钟的调控,其表达高峰出现在夜间。不同的生长发育时期,OsFBH1的表达水平不同。在播种后50天,OsFBH1出现表达高峰。激光共聚焦显微镜观察结果表明,OsFBH1所编码的蛋白质定位在细胞核中。3.超表达OsFBH1可以明显延迟拟南芥的开花时间。与野生型(wild-type,WT)相比,拟南芥OsFBH1超表达转基因株系的莲座叶数目显著增加,现蕾时间及第一朵花开放时间明显延迟。发育时期转换的试验结果表明,拟南芥OsFBH1的超表达转基因株系,使得拟南芥幼年叶和成年叶增加,延长了幼年营养生长期和成年营养生长期,从而延长了拟南芥从营养生长向生殖生长过渡的时间,最终引起拟南芥晚花。分子水平检测结果表明,OsFBH1表达上调,使促进拟南芥开花的关键基因,例如:Flowering Locus T(FT),Twin sister of FT,Suppressor of Overexpression of CONSTANS1(SOC1)的表达水平下调,从而延迟拟南芥开花。4.超表达OsFBH1可以明显的延迟水稻的开花期。与野生型(沈农9816,SN9816)相比,超表达OsFBH1水稻转基因株系开花期明显延迟。分子水平检测结果表明,OsFBH1表达上调,导致促进水稻开花的关键基因,例如:Heading Date 3a和Rice Flowering locus T1的表达水平明显降低,从而,延迟水稻开花时间。此外,超表达OsFBH1水稻转基因株系还表现为株高降低,水稻籽粒宽度变窄,分蘖数减少等多种生长发育异常的表型。5.超表达OsFBH3促进拟南芥开花。与WT相比,超表达OsFBH3拟南芥转基因株系表现为早花。莲座叶数目明显减少,现蕾时间和开花时间明显早于WT。发育时期转换的试验结果表明,OsFBH3没有影响幼年营养生长期向成年营养生长期的时期转换,主要是缩短了成年营养生长期,促进了营养生长向生殖生长的时期转换,进而导致拟南芥开花提前。RT-PCR的结果表明,在长日照和短日照条件下,拟南芥OsFBH3超表达转基因株系提高CONSTANS的表达。从而提高SOC1和FT的表达水平,促进了拟南芥开花。除了影响开花期外,超表达OsFBH3还导致拟南芥其它方面的生长发育异常,表现在株型变小,叶片和花变小,花苔变细,果荚变短,胚胎数目减少,败育胚胎数目增加等方面。6.OsFBH 的表达模式和亚细胞定位分析。荧光定量RT-PCR和GUS染色的研究结果表明,OsFBH3主要在水稻的叶片,叶鞘和茎干中表达;在不同时期OsFBH3的表达水平不同,在播种后50天左右,OsFBH3出现表达高峰。OsFBH3的表达受生物钟的调控,表达最高峰出现在夜间。激光共聚焦显微镜观察结果表明,OsFBH3所编码的蛋白质定位于细胞核。
[Abstract]:The flowering period plays a very important role in the development of rice (Oryza sativa) and the process of yield formation. The flowering period of rice is the interaction of endogenous and external environmental conditions, multi gene participation, and complex life process. Although Heading date 1 and Early heading date 1 have been preliminarily established as the key regulating factors However, the molecular mechanism of Rice Flowering Regulation is far from clear. With the help of bioinformatics, this project uses the sequence of the Flowering bHLHs (FBHs) gene family of the flowering time related gene of Arabidopsis (Arabidopsis thaniala) to select the homologous gene OsFBHs in rice. Using CRISPR-Cas9 gene editing technique, DNA recombination and rice transgenic technology, the overexpression of OFBVHs gene was created, and the transgenic rice or Arabidopsis thaliana was downregulated and the genes were knocked out of the gene. After observing the agronomic characters of the transgenic lines, the transformation of the OsFBH1 and OsFBH3 genes of the agronomic traits, such as the flowering period, was obtained. In this project, we used these transgenic lines as test materials, using fluorescence quantitative RT-PCR, yeast single (double) hybridization, chromatin immunoprecipitation (Chromatin Immunoprecipitation, ChIP) and so on. This project deeply studied the expression pattern of OsFBH, subcellular localization and regulation of the target gene of flowering period, and revealed that OsFBHs regulates rice. The molecular mechanism of flowering period is a theoretical basis for improving rice varieties at different growth stages, improving rice adaptability, yield and quality. The main results of this project are as follows: 1. the FBHs gene of rice OsFBHs and Arabidopsis thaliana are conserved and closely related. There are 4 members of the FBHs gene family in Arabidopsis (including: FBH1-4-). The code contains proteins with HLH conserved domains. 4 FBHs homologous genes (OsFBH1-4) in rice are closely related to FBHs affinity in Arabidopsis. OsFBH1 and OsFBH2 are closely related to FBH1 and FBH2 in Arabidopsis, and OsFBH3 and OsFBH4 are closely related to FBH3 and FBH4 in Arabidopsis. The results of localization analysis. The results of fluorescence quantitative RT-PCR and GUS staining showed that the expression level of OsFBH1 in rice leaves and Ye Qiaozhong was higher. The expression of.OsFBH1 in the root and the coleoptile tissues was also regulated by the biological clock. The expression peak appeared at night. The expression level of OsFBH1 was different. 50 days after sowing, OsFBH1 showed a peak expression. The results of laser confocal microscopy showed that the.3. overexpression of the protein encoded by OsFBH1 in the nucleus could significantly delay the flowering time of Arabidopsis. Compared with the wild type (wild-type, WT), the number of lotus leaves in the transgenic lines of OsFBH1 overexpressed in Arabidopsis was increased significantly, and the bud time and the first bud time were increased. The opening time of a flower was obviously delayed. The experimental results of the developmental period transformation showed that the overexpression transgenic line of Arabidopsis OsFBH1 made the young leaf of Arabidopsis and adult Ye Zengjia prolonged the vegetative growth period and the adult vegetative growth period, thus prolonging the time of the Arabidopsis thaliana transition from the camps to the reproductive growth. The results of molecular level detection showed that the expression of OsFBH1 was up-regulated, which made the key genes promoting the flowering of Arabidopsis, such as Flowering Locus T (FT), Twin sister of FT, Suppressor of Overexpression. Compared with the wild type (Shen Nong 9816, SN9816), the flowering period of the transgenic lines of OsFBH1 rice was obviously delayed. The results of molecular level detection showed that the expression of OsFBH1 was up, leading to the key genes promoting the flowering of rice, such as the expression level of Heading Date 3a and Rice Flowering locus T1, so that the rice flowering was delayed. In addition, the overexpression of OsFBH1 transgenic lines also showed that the height of the plant decreased, the width of the rice grain was narrowed, the number of tillers decreased and the number of.5. overexpressed OsFBH3 to promote the flowering of Arabidopsis. Compared with WT, the overexpression of OsFBH3 in the transgenic plants of Arabidopsis thaliana was shown to be early flowers. The experimental results of the flowering time, which was earlier than the WT. development period, showed that OsFBH3 did not affect the period of young vegetative growth period to the period of adult vegetative growth, which mainly shortened the adult vegetative growth period, promoted the period of vegetative growth to the period of reproductive growth, and then led to the result of early flowering of Arabidopsis.RT-PCR. Under the condition of long sunshine and short sunshine, the OsFBH3 overexpression of Arabidopsis thaliana can increase the expression of CONSTANS, thus improving the expression level of SOC1 and FT and promoting the flowering of Arabidopsis. In addition to the flowering period, the overexpression of OsFBH3 also leads to the growth and development of Arabidopsis, which shows the smaller plant type, the smaller leaves and flowers, and the flower moss. The expression pattern and subcellular location analysis of.6.OsFBH, the results of fluorescence quantitative RT-PCR and GUS staining showed that OsFBH3 was mainly expressed in the leaves, leaf sheath and stem of rice, and the expression level of OsFBH3 was different at different times, 50 days after sowing, OsF The expression of the expression peak of BH3 was regulated by the biological clock, and the peak expression appeared at night. The results of laser confocal microscopy showed that the protein encoded by OsFBH3 was located in the nucleus.
【学位授予单位】：沈阳农业大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：S511

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