【作者】 李远婷；

【导师】 安黎哲；

【作者基本信息】 兰州大学 ， 植物学， 2012， 博士

【摘要】 对于植物来说,重力代表了一个重要的环境信号,它影响了植物的生长和发育。向重力性是指植物能够感受重力的方向,使得根向下生长,茎向上生长。根的向重力性在拟南芥中已被广泛研究,但是向重力性的机理还没有被完全研究清楚。植物的向重力性、向光性和生长素调节器官发育对植物的形态建成很重要。人们通过对拟南芥向光性和生长素调节器官发育的研究发现二者都至少需要一个NPH3样基因,一个AGC激酶和一个生长素响应因子。NPH3样基因是一个大的基因家族,NPY基因属于这个家族。许多研究表明生长素响应因子(ARF7和ARF19)和AGC激酶(PID, WAG1, WAG2和D6PKs)对根的向重力性也起重要作用。因此本论文研究了NPY基因是否也参与调节根的向重力性。主要获得以下研究结果：(1)为了研究NPY基因在根里的表达特异性,通过GUS染色发现NPY1, NPY2, NPY3, NPY4, NPY5都在主根的根尖表达。NPY基因在根尖的表达揭示了这5个NPY基因很可能对根向重力性起作用。(2)为了研究NPY基因的缺失是否直接影响根的向重力性,通过分析所有npy单突变体,双突变体,三突变体,四突变体和五突变体的根向重力性的表型,发现npy单突变体和双突变体没有明显的向重力性缺陷；在十种npy三突变体里,除了npy1npy2npy4,其他的三突变体略有向重力性缺陷；在五种npy四突变体里,除了npy1npy2npy3npy4,其他的四突变体均比npy三突变体的向重力性缺陷严重,npy2npy3npy4npy5四突变体是所有四突变里向重力性缺陷最严重的；npy1npy2npy3npy4npy5五突变体缺失向重力性在所有突变体里最严重。这些表型分析揭示了拟南芥的5个NPY基因对根向重力性功能冗余。(3)生长素运输抑制剂(NPA)抑制野生型的根向重力性,促进野生型根毛生长。通过NPA处理npy2npy3npy4npy5四突变体,发现突变体的非向重力性程度略有减弱,根毛密度和长度也比野生型少。结果说明NPY基因减弱了NPA抑制向重力性的作用,增强了NPA促进根毛生长的作用,揭示了NPY基因与生长素运输紧密相关。(4)生长素在野生型根的静止中心和根冠小柱细胞积累。为了研究NPY基因的突变是否会改变生长素在根尖的积累,通过激光扫描共聚焦显微技术研究了生长素报道基因DR5::GFP在npyl npy2npy3npy4npy5五突变体的表达,结果显示生长素不仅在npy五突变体的静止中心和根冠小柱细胞积累,还在侧根冠细胞(LRC)异常积累。揭示了生长素在npy五突变体根表皮细胞的极性运输受到影响。(5)PIN2基因编码了一个生长素输出载体。为了研究NPY基因与PIN2基因的相互作用,本论文以npy2npy3npy4npy5为母本,pin2为父本进行杂交,获得的npy2npy3npy4npy5pin2五突变体的向重力性缺陷均比父本或母本严重。同时,外源生长素IAA和NAA导致npy2npy3npy4npy5pin2五突变体根尖弯向培养基外侧生长的表型也比父本或母本严重。揭示了NPY基因和PIN2基因在生长素调节根向重力性的遗传通路里具有协同作用。(6)生长素运输蛋白PIN2定位在根尖表皮细胞膜的上层。为了研究NPY基因是否影响PIN2的定位,通过激光扫描共聚焦显微技术研究了PIN2::GFP在npy1npy2npy3npy4npy5五突变体的表达,结果显示PIN2不仅在npy五突变体根表皮细胞膜的上层定位,还在表皮细胞膜的侧面定位。这种极性定位的改变很可能是造成生长素极性运输在npy五突变体里受到抑制的原因。综上所述,NPY基因在拟南芥根向重力性里发挥着重要作用。研究根向重力性对于阐明地球重力在生物进化进程中的作用和空间不同重力环境中发展植物栽培技术具有重要意义。本论文揭示了拟南芥使用了AGC激酶-NPH3样蛋白-ARF模型来调节根向重力性,为进一步研究拟南芥根向重力性的机制奠定了基础。更多还原

【Abstract】 Gravity is an important environment signal for plants, effecting its growth and development. Plants can sense the direction of gravity and orient their growth to ensure that roots are anchored in soil and that shoots grow upward. Gravitropism have been studied extensively using Arabidopsis genetics, but the exact mechanisms are not fully understood. Gravitropism, phototropism and auxin-regulated organogenesis are important for morphogenesis. It was reported that auxin-regulated organogenesis and phototropism used analogous mechanisms for which at least one NPH3-like gene, one AGC kinase, and one ARF were required. NPH3-like gene is a big gene family, which NPY genes are included. It was reported that root gravitropic responses required both auxin response factors (ARF7and ARF19) and AGC kinase (PID, WAG1, WAG2and D6PKs). This paper researched whether NPY genes were required for gravitropic responses. The main results were as follows:(1) The expression patterns of the NPY genes were shown by GUS staining. All of the five NPY genes were highly expressed in primary root tips, indicating that they may play a key role in Arabidopsis root gravitropism.(2) The single and double npy mutants did not display obvious gravitropism defects; all of the npy triple mutants except npyl npy2npy4had weak gravitropism defects; all of the npy quadruple mutants except npyl npy2npy3npy4had stronger gravitropism defects than the npy triple mutants. The npyl npy2npy3npy4npy5quintuple mutants showed the strongest gravitropic phenotypes, second is npy2npy3npy4npy5quadruple mutants. Systematic phenotypic analysis of npy mutant demonstrated that the five NPY genes all contributed to gravitropism redundancily.(3) Auxin transport inhibitor NPA inhibits gravitropism and promotes the growth of root hair. Our data showed that NPA slightly decreased the agravitropism in npy2npy3npy4npy5quadruple mutants and npy2npy3npy4npy5quadruple mutants were more resistant to NPA in length and dencity of root hair than wild type, indicating NPY genes deseased the inhibitor function of NPA to gravitropism and increased the promote function of NPA to root hair, which uncovered that NPY genes are related to auxin transportion.(4) Auxin is accumulated in quiescent center and columella cells in wild type. The expression pattern of auxin reporter DR5::GFP in npyl npy2npy3npy4npy5quintuple mutants was shown by confocal. DR5::GFP not only detected in quiescent center and columella cells, but also in the lateral root cap in npy quintuple mutants, indicating auxin transportion was affected in npy quintuple mutants.(5) PIN2gene encoded a polar-auxin-transport efflux. Our data indicated NPYs and PIN2had synergistic interactions since npy2npy3npy4npy5pin2quintuple mutants showed stronger phenotype than either pin2single mutant or npy2npy3npy4npy5quadruple mutant in both agravitropism and the phenotype of root tip bending out of the plates with exogenous IAA and NAA.(6) PIN2located in upper side of epidermis of root tip in wild type. Our data showed that there was no PIN2polarity in epidermis of root tip in npyl npy2npy3npy4npy5quintuple mutants, probably causing the restrain of the polar auxin transportion from root tip to elongation zone through epidermis.In summary, NPY genes played an essential role in root gravitropic responses in Arabidopsis. The research of gravitropism is very important to clarify the function of gravity to biological evolution and help developing cultivation techniques in different gravity environments. This paper uncovered that plants used an AGC kinase-NPH3-like protein-ARF module analogous to those in auxin-regulated organogenesis and phototropism to regulate plant root gravitropic responses, which providing foundation for further understanding the exact mechanisms of gravitropism.更多还原