拟南芥蛋白激酶PTI1-5在花粉管和根毛生长中的作用研究

发布时间：2017-12-27 10:18

本文关键词：拟南芥蛋白激酶PTI1-5在花粉管和根毛生长中的作用研究　出处：《中国农业大学》2017年博士论文　论文类型：学位论文

更多相关文章： 拟南芥 PTI1-5 蛋白激酶 花粉管 根毛

【摘要】：花粉管和根毛都是呈顶端生长的植物细胞,分别在植物有性生殖和水分营养吸收方面起重要作用,与农业生产密切相关。在植物开花时,由雄蕊花药产生的花粉粒被释放出来并传递到雌蕊的柱头上,经水合后由其营养细胞萌发出花粉管。花粉管进行顶端生长,侵入柱头细胞后经花粉管通道最后把其携带的两个精细胞传递到胚囊中完成双受精。花粉管生长是植物有性生殖过程中的一个重要环节,花粉管生长缺陷会导致雄性不育。根毛是由根尖表皮的生毛细胞(trichoblasts)发育而来,根毛的形成显著地增加了根表皮细胞的表面积,有助于根对土壤水分营养的吸收和与土壤微生物的相互作用,促进植物生长发育和有效地应对环境胁迫。此外,花粉管和根毛也是良好的研究细胞极性生长的模式材料。研究花粉管和根毛生长的分子遗传机制不仅可以了解植物细胞生长发育的调控机制,也可为农作物栽培和育种提供遗传学方面的理论参考。研究表明,花粉管和根毛的生长受到Ca2+、小G蛋白、细胞骨架和蛋白激酶等诸多因素的调控,但目前对花粉管和根毛生长的分子遗传机制的了解还很有限,尤其是相关蛋白激酶在花粉管和根毛生长过程中的作用仍知之甚少。我所在的研究组长期致力于植物雄配子体(即花粉)发育的分子遗传学研究,在前期工作中分离到了一个Ds转座子插入引起的雄配子体缺陷突变体kd640,初步的分析结果显示,Ds插入到拟南芥Pto-interacting 1(PTI1)蛋白激酶家族成员PTI1-5(At2g41970)基因中,严重影响花粉管的生长,导致配子体型雄性不育。本论文是在原有研究基础上对pti1-5突变体进行详细地鉴定分析,并研究PTI1-5在花粉管生长发育中的作用。后来的研究结果显示,PTI1-5不但在花粉管中表达,而且也在根毛中有较高的表达。因此,又进一步分析了PTI1-5在根毛生长发育过程中的作用。pti1-5是一个从拟南芥基因陷阱(gene-trap)和增强自陷阱(enhancer-trap)Ds插入突变体库中筛选分离得到的雄配子体缺陷突变体。遗传分析表明,PTI1-5基因的突变导致雄配子体的传递完全丧失,不能产生纯合pti1-5突变体植株。表型分析发现,突变体花粉萌发后即发生爆裂,严重影响花粉管的正常生长,表明PTI1-5在花粉管生长中起重要作用。定量Real-time PCR和启动子分析结果显示,PTI1-5基因主要在花粉、花粉管、根和根毛中表达。利用花粉管特异启动子启动PTI1-5基因在pti1-5突变体花粉和花粉管中特异表达,能恢复突变体的雄配子体功能,并能获得纯合pti1-5突变体植株,结果发现Pti1-5的突变还会影响根毛的生长,表明PTI1-5不仅在花粉管生长发育中起重要作用,而且在根毛生长发育中发挥着重要的作用。PTI1-5 编码一个预测为胞质类受体蛋白激酶(receptor-like cytoplasmic protein kinase,RLCK)的蛋白质,是拟南芥PTI1激酶亚家族中的一个成员。PTI1激酶属于类受体胞质激酶RLCK VIII亚家族,共有11个成员。PTI1-5蛋白主要定位于正在生长的花粉管和根毛的细胞质和顶端质膜上。酵母双杂交、荧光素酶互补成像实验和免疫共沉淀实验显示,PTI1-5能和Oxidative signal-inducible 1(OXI1)及与其同源关系最近的蛋白激酶AGC2-2相互作用。磷酸化实验表明PTI1-5在体外能被OXI1磷酸化。已有研究表明OXI1主要在根毛中表达,而且oxi1突变体主要是影响根毛的生长,不影响花粉管的生长。因此,PTI1-5可能是通过与OXI1互作参与相关的信号转导,从而调控根毛的生长发育。此外,点突变实验结果显示,预测的磷酸化位点T239和T244对花粉萌发和花粉管的生长是十分重要的,这两个位点突变会严重影响花粉管的萌发与生长。但这两个位点是否是真的磷酸化位点和是否与OXI1对PTI1-5的磷酸化作用相关还不清楚。总而言之,本论文的研究结果显示,PTI1激酶家族成员PTI1-5在花粉管和根毛的生长中起重要作用。在根毛的生长过程中,PTI1-5可能是通过与OXI1互作参与相关的信号传递,从而调控根毛的生长。这些研究结果可为更加深入地了解和研究PTI1家族成员在植物生长发育中的作用提供参考信息,也可为进一步研究花粉和根毛生长的分子机制提供新的线索和思路。
[Abstract]:The pollen tube and root hairs were the top growth of plant cells, which plays an important role in plant sexual reproduction and moisture absorption of nutrients, and is closely related to agricultural production. During the flowering of plants, the pollen grains produced by the stamen anther are released and transferred to the stigma of the pistil, and the pollen tube is germinated by its nutrient cells after hydration. The pollen tube grows at the top, invades the stigma cells and passes the two sperm cells carried by the pollen tube channel to the embryo sac to complete the double fertilization. Pollen tube growth is an important link in the process of sexual reproduction in plants, and the growth defects of pollen tube can lead to male infertility. Hair from the apical epidermal hair cell (trichoblasts) development, root hair formation significantly increased the root epidermal cell surface area, can interact with the root on soil moisture and nutrient absorption and soil microorganism, promoting plant growth and development and effectively responding to environmental stress. In addition, mode of material research is also good for pollen tube and root hair cell polar growth. The molecular genetic mechanism of pollen tube growth and root hair can not only understand the regulation mechanism of the growth and development of plant cells, but also can provide a theoretical reference for the genetics of crop cultivation and breeding. The research shows that the regulation of pollen tube growth and root hairs by Ca2+ and small G protein, cytoskeleton and protein kinase and many other factors, but the molecular genetic mechanism of the pollen tube growth and root hairs are still poorly understood, especially the growth associated protein kinase function in the process of pollen tube and root hairs are still poorly understood. Our research group is committed to long-term plant male gametophyte (i.e., pollen) molecular genetics research and development, in the previous work, we isolated a Ds transposon insertion caused by male gametophyte mutants kd640, preliminary results of the analysis show that the Ds into Arabidopsis Pto-interacting 1 (PTI1) protein kinase family members PTI1-5 (At2g41970) gene, seriously affected the growth of pollen tube, resulting in gametophytic male sterility. This paper is based on the original research on the pti1-5 mutants in detail, and studies the role of PTI1-5 in the growth and development of pollen tube. The results show that PTI1-5 not only expressed in the pollen tube, but also has a high expression in root hairs. Therefore, further analysis of PTI1-5 in root hair development process. Pti1-5 is a male gametophyte defective mutant screened from Arabidopsis gene trap (gene-trap) and enhanced self trap (enhancer-trap) Ds insertion mutant library. Genetic analysis showed that the mutation of PTI1-5 gene resulted in complete loss of male gametophyte transmission, and no homozygous pti1-5 mutant plants could be produced. Phenotypic analysis showed that the mutant pollen burst after germination, which seriously affected the normal growth of pollen tube, indicating that PTI1-5 played an important role in the growth of pollen tube. Quantitative Real-time PCR and promoter analysis showed that PTI1-5 gene mainly expressed in pollen, pollen, and root hair. The pollen specific promoter of PTI1-5 gene promoter in pti1-5 mutant pollen and pollen tube specifically expressed in the male gametophyte function can restore the mutant, and can obtain homozygous pti1-5 mutant, the mutation was found in root hairs also affect Pti1-5 growth, show that PTI1-5 not only plays an important role in pollen tube growth and development, but also in root hair growth play an important role in. PTI1-5 encodes a protein predicted to be receptor-like cytoplasmic protein kinase (RLCK), a member of the PTI1 kinase subfamily of Arabidopsis thaliana. PTI1 kinase belongs to the receptor like cytoplasmic kinase RLCK VIII subfamily, with a total of 11 members. PTI1-5 protein was mainly localized in the growing pollen tube and root hairs on the apical membrane and cytoplasm. Yeast two hybrid, luciferase complementary imaging and co immunoprecipitation experiments showed that PTI1-5 could interact with Oxidative signal-inducible 1 (OXI1) and its closest protein kinase AGC2-2. The experiment of phosphorylation showed that PTI1-5 could be phosphorylated by OXI1 in vitro. Studies have shown that OXI1 is mainly expressed in root hairs, and oxi1 mutant mainly affect the root hair growth, does not affect the growth of pollen tube. Therefore, PTI1-5 may be through interaction with OXI1 involved in signal transduction, thereby regulating the growth and development of root hairs. In addition, point mutation experiments showed that the predicted phosphorylation sites T239 and T244 are important for pollen germination and pollen tube growth. These two loci will seriously affect the germination and growth of pollen tube. However, it is not clear whether these two loci are true phosphorylation sites and whether OXI1 is phosphorylated with PTI1-5. In a word, the research results show that PTI1 kinase family members PTI1-5 play an important role in the growth of root hairs and pollen tube. In the process of growth of root hairs in PTI1-5 may be through interaction with OXI1 involved in signal transmission, and growth regulation of root hairs. These results can provide more in-depth understanding and research of PTI1 family members in plant growth and development in the role of providing reference information, but also provide new clues and ideas for the further research of the molecular mechanism of pollen and hair growth.
【学位授予单位】：中国农业大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：Q945

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