本文關鍵詞：擬南芥蛋白激酶PTI1-5在花粉管和根毛生長中的作用研究　出處：《中國農(nóng)業(yè)大學》2017年博士論文　論文類型：學位論文

  更多相關文章： 擬南芥 PTI1-5 蛋白激酶 花粉管 根毛

【摘要】：花粉管和根毛都是呈頂端生長的植物細胞,分別在植物有性生殖和水分營養(yǎng)吸收方面起重要作用,與農(nóng)業(yè)生產(chǎn)密切相關。在植物開花時,由雄蕊花藥產(chǎn)生的花粉粒被釋放出來并傳遞到雌蕊的柱頭上,經(jīng)水合后由其營養(yǎng)細胞萌發(fā)出花粉管�；ǚ酃苓M行頂端生長,侵入柱頭細胞后經(jīng)花粉管通道最后把其攜帶的兩個精細胞傳遞到胚囊中完成雙受精。花粉管生長是植物有性生殖過程中的一個重要環(huán)節(jié),花粉管生長缺陷會導致雄性不育。根毛是由根尖表皮的生毛細胞(trichoblasts)發(fā)育而來,根毛的形成顯著地增加了根表皮細胞的表面積,有助于根對土壤水分營養(yǎng)的吸收和與土壤微生物的相互作用,促進植物生長發(fā)育和有效地應對環(huán)境脅迫。此外,花粉管和根毛也是良好的研究細胞極性生長的模式材料。研究花粉管和根毛生長的分子遺傳機制不僅可以了解植物細胞生長發(fā)育的調(diào)控機制,也可為農(nóng)作物栽培和育種提供遺傳學方面的理論參考。研究表明,花粉管和根毛的生長受到Ca2+、小G蛋白、細胞骨架和蛋白激酶等諸多因素的調(diào)控,但目前對花粉管和根毛生長的分子遺傳機制的了解還很有限,尤其是相關蛋白激酶在花粉管和根毛生長過程中的作用仍知之甚少。我所在的研究組長期致力于植物雄配子體(即花粉)發(fā)育的分子遺傳學研究,在前期工作中分離到了一個Ds轉(zhuǎn)座子插入引起的雄配子體缺陷突變體kd640,初步的分析結(jié)果顯示,Ds插入到擬南芥Pto-interacting 1(PTI1)蛋白激酶家族成員PTI1-5(At2g41970)基因中,嚴重影響花粉管的生長,導致配子體型雄性不育。本論文是在原有研究基礎上對pti1-5突變體進行詳細地鑒定分析,并研究PTI1-5在花粉管生長發(fā)育中的作用。后來的研究結(jié)果顯示,PTI1-5不但在花粉管中表達,而且也在根毛中有較高的表達。因此,又進一步分析了PTI1-5在根毛生長發(fā)育過程中的作用。pti1-5是一個從擬南芥基因陷阱(gene-trap)和增強自陷阱(enhancer-trap)Ds插入突變體庫中篩選分離得到的雄配子體缺陷突變體。遺傳分析表明,PTI1-5基因的突變導致雄配子體的傳遞完全喪失,不能產(chǎn)生純合pti1-5突變體植株。表型分析發(fā)現(xiàn),突變體花粉萌發(fā)后即發(fā)生爆裂,嚴重影響花粉管的正常生長,表明PTI1-5在花粉管生長中起重要作用。定量Real-time PCR和啟動子分析結(jié)果顯示,PTI1-5基因主要在花粉、花粉管、根和根毛中表達。利用花粉管特異啟動子啟動PTI1-5基因在pti1-5突變體花粉和花粉管中特異表達,能恢復突變體的雄配子體功能,并能獲得純合pti1-5突變體植株,結(jié)果發(fā)現(xiàn)Pti1-5的突變還會影響根毛的生長,表明PTI1-5不僅在花粉管生長發(fā)育中起重要作用,而且在根毛生長發(fā)育中發(fā)揮著重要的作用。PTI1-5 編碼一個預測為胞質(zhì)類受體蛋白激酶(receptor-like cytoplasmic protein kinase,RLCK)的蛋白質(zhì),是擬南芥PTI1激酶亞家族中的一個成員。PTI1激酶屬于類受體胞質(zhì)激酶RLCK VIII亞家族,共有11個成員。PTI1-5蛋白主要定位于正在生長的花粉管和根毛的細胞質(zhì)和頂端質(zhì)膜上。酵母雙雜交、熒光素酶互補成像實驗和免疫共沉淀實驗顯示,PTI1-5能和Oxidative signal-inducible 1(OXI1)及與其同源關系最近的蛋白激酶AGC2-2相互作用。磷酸化實驗表明PTI1-5在體外能被OXI1磷酸化。已有研究表明OXI1主要在根毛中表達,而且oxi1突變體主要是影響根毛的生長,不影響花粉管的生長。因此,PTI1-5可能是通過與OXI1互作參與相關的信號轉(zhuǎn)導,從而調(diào)控根毛的生長發(fā)育。此外,點突變實驗結(jié)果顯示,預測的磷酸化位點T239和T244對花粉萌發(fā)和花粉管的生長是十分重要的,這兩個位點突變會嚴重影響花粉管的萌發(fā)與生長。但這兩個位點是否是真的磷酸化位點和是否與OXI1對PTI1-5的磷酸化作用相關還不清楚�？偠灾�,本論文的研究結(jié)果顯示,PTI1激酶家族成員PTI1-5在花粉管和根毛的生長中起重要作用。在根毛的生長過程中,PTI1-5可能是通過與OXI1互作參與相關的信號傳遞,從而調(diào)控根毛的生長。這些研究結(jié)果可為更加深入地了解和研究PTI1家族成員在植物生長發(fā)育中的作用提供參考信息,也可為進一步研究花粉和根毛生長的分子機制提供新的線索和思路。
[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.
【學位授予單位】：中國農(nóng)業(yè)大學
【學位級別】：博士
【學位授予年份】：2017
【分類號】：Q945

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本文編號：1341311