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摘要：RING（Really Interesting New Gene）类E3连接酶是含有锌指结构域，能够特异性识别底物并将其与泛素分子相连接的一类蛋白。被泛素化的底物通常会在 26S 蛋白酶体中被降解。已知这种泛素-蛋白酶体途径在水稻抗逆、生长发育等过程中起重要作用，但其在生殖发育中的作用还很不清楚。本文通过RNA-Seq 数据分析及通过酵母双杂交筛选cDNA文库对水稻早期花药发育相关的 E3连接酶进行筛选与验证。在之前的工作中本实验室找到了一个调控水稻早期花药发育的重要转录调控因子 MPL（MICROSPOROCYTELESS） ，MPL基因突变后水稻小孢子母细胞丧失，不能形成花粉。为了寻找调控MPL的E3泛素连接酶， 本研究通过分析RNA-Seq数据获取了MPL调控的编码含RING结构域蛋白的基因共22个。运用酵母双杂交技术验证了其中15 个与MPL间的相互作用，结果表明这些含有 RING 结构域蛋白均不与 MPL 相互作用。接着本研究以 MPL 作为诱饵，筛选水稻幼穗的cDNA 文库，获得到了 8个编码含RING结构域的蛋白可能与MPL相互作用，相关验证工作正在进行。这些RING 类 E3 连接酶可能通过在蛋白水平上调控 MPL实现对水稻早期花药发育的调控。该研究为研究RING 类E3连接酶在生殖发育中的作用以及 MPL的调控提供了重要线索。 25299
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关键词：水稻；RING 类E3连接酶；花药发育；MPL；酵母双杂交
Screening for RING Type E3 Ligase Involved in Rice Early Anther Development
Abstract： RING (Really Interesting New Gene) type E3 ligase is a protein family containing zinc finger
domain that is capable of specifically recognizing a substrate and linking it to ubiquitin molecules. The
ubiquitinated substrate is then usually degraded in the 26S proteasome. It is known that
ubiquitin-proteasome pathway plays an important role in rice resistance to stresses, plant growth and
development. However, the role of ubiquitin-proteasome pathway in rice reproductive development
remains largely unclear. In this thesis, I screened E3 ligases involved in rice early anther development by
RNA-Seq data analysis and yeast two-hybrid. In our previous work，we identified a transcriptional
regulator MPL (MICROSPOROCYTELESS) plays an essential role in rice early anther development.
Disruption of MPL caused defects in formation of microspores and thus no pollen production. In order to
find E3 ligases that regulate MPL, I obtained 22 genes encoding proteins containing RING domain by
analyzing RNA-Seq data. I tested the interactions between MPL and 15 of the 22 E3 ligases by yeast
two-hybrid assays. No E3 ligases were found to interact with MPL. Then I used MPL as a bait to screen the
rice early anther cDNA library for E3 ligases involved in rice early anther development. I obtained eight
genes encoding proteins containing RING domain. The confirmation of interaction between these RING
protein with MPL is underway. These results provide valuable clues to identify novel RING-type E3 ligases
in early anther development and the molecular mechanisms of MPL regulation.
Key words: Rice; RING-type E3 ligase; Anther development; MPL; Yeast two-hybrid
摘要„1
关键词1
Abstract„1
Key words2
第一章 文献综述„2
1.1 水稻早期花药发育„2
1.1.1 水稻花药细胞分化过程2
1.1.2 水稻花药发育早期的调控„2
1.2 RING类E3泛素连接酶3
1.2.1 作用机理3
1.2.2 转录因子调控与自动反馈循环„3