兰州大学机构库 >生命科学学院
OsLPL2的功能初探及一个拟南芥topp4-1恢复突变体的鉴定
Alternative TitleFunctional Analysis of OsLPL2 and Identification of topp4-1 Revertant Mutant in Arabidopsis
王育川
Thesis Advisor侯岁稳
2016-06-01
Degree Grantor兰州大学
Place of Conferral兰州
Degree Name硕士
Keyword表皮细胞 OsLPL2 微丝 成核调控 TOPP4 恢复突变体
Abstract微丝骨架在植物表皮细胞形态建成的过程中扮演了至关重要的角色,其结构及排列方式的异常会导致一系列缺陷表型。当前对微丝的研究多集中于拟南芥ROP-SCAR/WAVE-ARP2/3成核信号通路,被小GTPase激活的SCAR/WAVE复合体(PIR/NAP125/HSPC300/ABI/SCAR)可以激活调控下游ARP2/3起始微丝成核。但在水稻中,微丝调控机制仍不明确。本实验室前期筛选到一个单基因隐性控制的突变体lpl2-1(less pronounced lobe epidermal cell 2-1),表现为表皮细胞边缘突出变平滑。突变体为Os03g05020(OsLPL2)转录提前终止。BLAST表明OsLPL2编码了拟南芥PIR同源蛋白。OsLPL2在高等植物中高度的氨基酸序列相似性预示着其功能可能存在一定的相似性。由于AtPIR与AtNAP125可以直接互作,我们订购了AtNAP125在水稻中的同源基因T-DNA突变体lpl3-1。该突变体表型与lpl2-1相似。为了进一步研究OsLPL2在水稻微丝调控中具体的功能,我们用酵母双杂探究水稻中是否存在与拟南芥相似的作用机制。结果表明,OsLPL2可与OsLPL3互作,结构域分段互作实验证明互作区段位于N端的PH区域;无论是OsLPL2还是OsLPL3均与OsLPL1(与AtHSPC300同源)无互作。进一步研究表明,OsLPL2未能与拟南芥ROP2同源蛋白OsRAC5、OsRAC6和OsRAC2互作。结合实验室之前结果,可证明水稻中存在与拟南芥中相似的微丝调控机制。 另外,我们从本实验室激活标签法构建的topp4-1恢复突变体库中筛选到一系列topp4-1表型恢复株系。本实验主要以5067-8#为研究对象,发现该株系在株高、叶形、果荚及表皮扁平细胞等方面都有恢复。测序并确定激活标签插入位点位于1号染色体。对插入位点附近10 kb的基因进行相对表达量的检测,以确定真正起恢复作用的基因。通过该研究,为进一步研究Ⅰ型磷酸酶TOPP4在植物生长发育中所起到的重要作用提供了基础材料。
Other AbstractActin cytoskeleton plays a critical role in plant epidermal cells during morphogenesis. Abnormal actin will result in a series of deficient phenotype. In Arabidopsis, current researches of actin nucleation are focusing on ROP-SCAR/WAVE-ARP2/3 signaling pathway. The SCAR/WAVE complex (PIR/NAP125/HSPC300/ABI/SCAR) is a effector that convert small GTPase signals into defined ARP2/3 activation responses. In rice, the regulatory mechanism of actin is still unknown. In previous study, lpl2-1 (less pronounced lobe epidermal cell 2-1), a mutant which is controlled by the recessive heredity of single gene of autosomes, have been identified. lpl2-1 shows epidermal pavement cells with smooth marginal lobes. The excalation of Os03g05020 (OsLPL2) leads to premature transcription termination. We run BLAST and find that OsLPL2 encodes a putative protein homologous to AtPIR. OsLPL2 shares high degree of amino acid similarity with its homologous proteins in higher plants which indicates that it may play a conservative role in evolution. Since AtPIR can directly interact with AtNAP125, we then order a rice mutant lpl3-1, whose mutant gene (OsLPL3) homologues to AtNAP125. lpl3-1 exhibits a phenotype similar to lpl2-1. To further investigate the possible function of OsLPL2 in actin regulation, yeast two-hybrid has been used to explore whether a similar mechanism of actin exists in rice. The results show that OsLPL2 can directly interact with OsLPL3 and the PH domain of OsLPL2 in the N-terminal, which plays a role in interaction. In contrast, neither OsLPL2 nor OsLPL3 can interact with OsLPL1 (homologues to AtHSPC300). We also demonstrate that OsLPL2 fails to interact with proteins (such as OsRAC5, OsRAC6 and OsRAC2) homologues to AtROP2. Together with previous results, we prove that regulatory mechanism of actin exists in rice which similar to Arabidopsis. In addition, we identify a series of revertant mutants by screening an activation tagging mutagenized topp4-1 population. In this study, we characterize the phenotypes of revertant mutant 5067-8#. It can rescue phenotype defects of topp4-1 in height, leaf shape, silique, pavement cell and so on. The T-DNA insertion site is located on chromosome 1. Relative expression of candidate gene has been detected to determine the true gene which can function in 5067-8#. This study provides basic materials for the important role of type-one phosphatase (TOPP4) during plant growth and development.
URL查看原文
Language中文
Document Type学位论文
Identifierhttps://ir.lzu.edu.cn/handle/262010/221906
Collection生命科学学院
Recommended Citation
GB/T 7714
王育川. OsLPL2的功能初探及一个拟南芥topp4-1恢复突变体的鉴定[D]. 兰州. 兰州大学,2016.
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