Molecular, Biochemical, and Structural Studies of the Mechanism of S-RNase-Based Self-Incompatibility in Petunia

矮牵牛基于 S-RNase 的自交不亲和机制的分子、生化和结构研究

基本信息

批准号：
1645557
负责人：
Teh-hui Kao
金额：
$ 68万
依托单位：
Pennsylvania State Univ University Park
依托单位国家：
美国
项目类别：
Continuing Grant
财政年份：
2017
资助国家：
美国
起止时间：
2017-03-15 至 2023-02-28
项目状态：
已结题

来源：
https://www.nsf.gov/awardsearch/showAward?AWD_ID=1645557&HistoricalAwards=false
关键词：
Molecular Biochemical Structural Studies Mechanism

项目摘要

Most flowering plants produce flowers with the male reproductive organ, the anther, and female reproductive organ, the pistil, located in close proximity. This arrangement would allow the pollen produced by the anther to land on the top of the pistil to result in self-pollination and consequent inbreeding. Inbreeding is deleterious to any organism, as it causes reduced fitness in progeny. Flowering plants have adopted self-incompatibility (SI), which allows the pistil to reject self-pollen to prevent inbreeding, but accept non-self pollen to promote outcrossing. Since Charles Darwin first documented this phenomenon in a monograph published in 1876, SI has attracted the interest of a wide spectrum of biologists. The PI has identified a pistil protein and 17 pollen proteins that are key players in mediating SI in Petunia inflata, a wild relative of garden petunia. In this project, the PI and his collaborator will use a combination of molecular, biochemical, genetic, genomic, and structural approaches to determine how the 17 pollen proteins and a single pistil protein work together to allow the pistil to specifically reject self-pollen. The results have wider implications for the study of many other biological systems employing self/non-self recognition. The genes for these proteins could be used to facilitate hybrid-seed production, which, if accomplished, would provide tremendous agronomic benefits. The PI will engage graduate and undergraduate students in this project to prepare them for future research careers, and will offer summer research opportunity for high school students through Penn State's Upward Bound Math and Science Program. In S-RNase-based self-incompatibility in Petunia inflata, the polymorphic S-locus controls the outcome of pollination; matching of the pollen S-haplotype with either S-haplotype of the pistil results in growth inhibition of pollen tubes. The PI has shown that the S-RNase gene controls pistil specificity and identified 17 S-locus F-box (SLF) genes in S2-haplotype that collectively control pollen specificity. In this project, the PI will complete the determination of 187 interaction relationships between all 17 SLF proteins and 11 allelic variants of S-RNase. These results will be used to study the biochemical basis of differential interactions between SLF proteins and S-RNases, and select SLF/S-RNase pairs for X-ray structural study of the interactions. Bacterial Artificial Chromosome (BAC) clones collectively containing the S-RNase and 17 SLF genes have been isolated, sequenced, and assembled, totaling ~3.1 Mbp. To fill gaps in the S-locus sequence, additional BAC clones will be isolated and sequenced. The PI has shown that all 17 SLF proteins form similar SCF complexes, but the Cullin1 and Skp1 components (PiCUL1-P and PiSSK1) are pollen-specific. The effect of knockout and suppressed expression of PiCUL1-P and PiSSK1 will be examined, and the X-ray structure of the SCFSLF complex will be determined. The PI has found that SLF proteins themselves are subject to ubiquitin-mediated degradation by the 26S proteasome, and identified proteins that may be involved in regulating the dynamics of the SCFSLF complex. The role of these proteins will be examined. All X-ray crystallization experiments will be performed in the lab of the collaborator.

大多数开花植物与雄性生殖器官，花药和雌性生殖器官The pistil产生花朵，位于近距离附近。这种布置将使花药产生的花粉能够降落在雌蕊的顶部，从而导致自花和随之而来的近交。近亲繁殖对任何生物体都是有害的，因为它会降低后代的健身性。开花植物采用了自我兼容（SI），这使雌蕊能够拒绝自花胶以防止近亲繁殖，但接受非自身的花粉来促进脱落。自查尔斯·达尔文（Charles Darwin）在1876年出版的一本专着中首次记录了这一现象以来，SI吸引了广泛的生物学家的兴趣。 PI已经确定了一种雌蕊蛋白和17种花粉蛋白，这些蛋白是在花园矮牵牛的野生亲戚petunia forda中介导Si的关键参与者。在这个项目中，PI和他的合作者将使用分子，生化，遗传，基因组和结构方法的结合，以确定17种花粉蛋白和单个雌蕊蛋白如何共同起作用，以使雌蕊特别拒绝自我胶片。结果对采用自我识别的许多其他生物系统的研究具有更大的影响。这些蛋白质的基因可用于促进杂交种子生产，如果完成，将提供巨大的农艺益处。 PI将吸引研究生和本科生参与该项目，以为未来的研究职业做准备，并通过宾夕法尼亚州立大学的高中界数学和科学计划为高中生提供夏季的研究机会。在基于S-RNase的矮牵牛足球中的自我兼容性中，多态S-cocus控制着授粉的结果。花粉s-光型与雌蕊的任何S-型型的匹配会导致花粉管的生长抑制。 PI表明，S-RNase基因控制雌蕊的特异性，并在S2-Haplotype中鉴定出17 s-cocus F-box（SLF）基因，这些基因集体控制花粉特异性。在该项目中，PI将完成对所有17个SLF蛋白与S-RNase的11个等位基因变体之间187个相互作用关系的确定。这些结果将用于研究SLF蛋白与S-RNase之间差异相互作用的生化基础，并选择SLF/S-RNase Pairs进行相互作用的X射线结构研究。细菌人造染色体（BAC）克隆共同含有S-RNase和17个SLF基因，已分离，测序和组装，总计约3.1 Mbp。为了填补S-Ocus序列中的间隙，将分离并测序其他BAC克隆。 PI表明，所有17种SLF蛋白都形成相似的SCF复合物，但是Cullin1和SKP1成分（PICUL1-P和PISSK1）都是花粉特异性的。将检查敲除和抑制pICUL1-P和PISSK1表达的影响，并将确定SCFSLF复合物的X射线结构。 PI发现，SLF蛋白本身受26S蛋白酶体的泛素介导的降解，并鉴定出可能参与调节SCFSLF复合物的动力学的蛋白质。将检查这些蛋白质的作用。所有X射线结晶实验将在合作者的实验室中进行。

项目成果

期刊论文数量（5）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Use of Domain-Swapping to Identify Candidate Amino Acids Involved in Differential Interactions between Two Allelic Variants of Type-1 S-Locus F-Box Protein and S3-RNase in Petunia inflata

DOI：
10.1093/pcp/pcx176
发表时间：
2018-02
期刊：
Plant and Cell Physiology
影响因子：
4.9
作者：
Lihua Wu;Justin S. Williams;Ning Wang;Wasi A Khatri;Daniele San Rom[REPLACEMENT CHARACTER]n;T. Kao
通讯作者：
Lihua Wu;Justin S. Williams;Ning Wang;Wasi A Khatri;Daniele San Rom[REPLACEMENT CHARACTER]n;T. Kao

S-Locus F-Box Proteins Are Solely Responsible for S-RNase-Based Self-Incompatibility of Petunia Pollen