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摘要：[目的]水稻是重要的粮食作物，水稻穗型与产量紧密相关。研究控制水稻穗型的基因功能以及发掘新的功能基因对提高水稻产量有重要意义。APO1与APO2蛋白可以直接相互作用并共同调控水稻幼穗分化。APO1是水稻的F-box蛋白，介导底物蛋白质的泛素化降解。本研究分析了APO1、APO2/RFL的功能，试图明确APO1和APO2/RFL的互作蛋白，以及构建与APO1和APO2/RFL互作基因的突变体。[方法]利用已有的apo1和apo2/rfl突变体，观察突变表型。并将mcherry蛋白基因与APO1连接，GFP蛋白基因与APO2/RFL连接，分别导入apo1和apo2/rfl突变体，观察表型恢复状况。提取apo1突变体和野生型水稻的幼穗蛋白，进行iTRAQ分析，获得差异表达蛋白质。后进行酵母双杂筛选，明确互作蛋白。利用CRISPR/Cas9技术构建互作基因突变体。[结果]与野生型水稻相比，apo1和apo2/rfl突变体的穗形明显变小，二次枝梗数也明显减小。apo2/rfl突变体导入gRFL:GFP以后，穗型出现一定程度的恢复。apo1突变体导入gAPO1：mchery后，APO1基因出现超表达，穗型较野生型明显变大。iTRAQ分析发现apo1与野生型的幼穗有279个蛋白表达存在显著差异，包括细胞分裂、分化与代谢等多方面的蛋白。酵母双杂筛选得到了与APO1和APO2/RFL蛋白直接互作的蛋白质。CRISPR/Cas9技术构建了多个突变体。 [结论]明确了APO1和APO2/RFL基因在蛋白层面对水稻穗分化的影响，为下一步了解它们的互作机制奠定基础。36602
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关键词：水稻；APO1；APO2/RFL；iTRAQ；酵母双杂；CRISPR/Cas9
The construct of the mutants interact with ABERRANT PANICLE ORGANIZATION 1 （APO1）/ABERRANT PANICLE ORGANIZATION 2 （APO2/RFL）of rice and the analyze of the function
Abstract: [Objectives] rice is an important crop. The shape of spike has a tight connection to the production. With which, do research about the genes of rice spike and dig out new function gene so that improve the production has a good meaning. The proteins of APO1 and APO2/RFL could interact with each other and cooperate to control the dedifferentiation of young spike of rice. APO1 is F-box protein of rice, mediate substrate protein ubiquitination degradation. The research analyzes the function of APO1 and APO2/RFL, try to make it clear about the substrate protein which APO1 degradation. At the same time, construct the mutants of the genes interact with APO1 and APO2/RFL. [Methods] make use of apo1 and apo2/rfl mutants have gained. First, observe the phenotypes compared with wild type. And connect the gene of mchery protein with APO1,connect the gene of GFP with APO2/RFL,turned them into apo1 and apo2/rfl mutant respectively. Observe the phenotype whether it recovers. Then, extract the proteins of young spikes of apo1 mutant and wild type, analyze with the technology of iTRAQ. Then filter by the technology of yeast two hybrid, so that gain the proteins interact with each other. Then we use the technology of CRISPR/Cas9 to construct the mutants of the genes interact with APO1 and APO2/RFL. [Results] Compared with wild type, the shape of spike of apo1 mutant was obviously smaller, the amount of secondary branches was also less. When gRFL:GFP turned into apo2/rfl mutant, the phenotype of spike was recovered to a certain extent. When gAPO1:mchery turned into apo1 mutant, the APO1 gene was overexpressed, with the phenotype of spike become bigger. The analyze of iTRAQ found that there are 279 proteins express with obvious difference, including cell pision, cell dedifferentiation, and metabolism, and so on. We also did Yeast two hybrid, to find the proteins which gene APO1 and APO2/RFL controls directly. The technology of CRISPR/Cas9 has constructed some mutants. [Conclusions] We demonstrated how APO1 and APO2/RFL have an influence at rice spike dedifferentiation at the angle of protein, which makes a base on the research on how APO1 and APO2/RFL interaction.
Keywords: rice; APO1; APO2/RFL; iTRAQ; Yeast two hybrid;CRISPR/Cas9
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