【作者】 王金凤；

【导师】 范云六；

【作者基本信息】 中国农业科学院 ， 生物化学与分子生物学， 2008， 博士

【摘要】 本论文第一部分工作是关于一个影响胚胎发育的AtFEM基因的研究。植物中精确控制基因的表达对于胚胎和胚乳的发育是非常关键的。目前已经发现,超过250个基因的突变可以导致胚胎发育异常(或致死),这些基因的正常表达对于胚胎的正常发育是必需的,对胚胎发育的影响不尽相同。已知的引起胚胎发育缺陷的基因编码执行不同功能的蛋白,包括代谢、细胞生长、转录、蛋白命运、蛋白转运和受阻以及植物防御。在拟南芥中, F-box蛋白有700个以上,通过参与SCF复合体介导底物蛋白降解。遗传分析表明,F-box蛋白在许多信号传导途径中发挥作用。关于F-box蛋白参与胚胎和胚乳发育已经有一些报道。F-box蛋白TIR1和AFB在胚胎形成过程中调节生长素的反应,tir1-1 afb2-1 afb3-1突变体的胚胎在早期发育异常。F-box蛋白AtSFL61和AtSFL70参与胚胎或雌配子体的发育,AtSFL61突变引起胚胎败育,突变体胚胎细胞不正常分裂;atsfl70/+杂合突变体胚珠种子败育率为50%,由雌配子体致死所引起。本论文中对一个F-BOX EMBRYO LETHAL (FEM)基因进行研究,预测该基因编码的蛋白是一个参与细胞周期的F-box蛋白,Atfem的T-DNA插入突变可以引起隐性胚胎致死表型。自交的杂合体植株产生几乎25%的不正常种子,这些种子最终萎缩败育。突变体的胚胎大部分停滞在晚球形期,其中有的败育胚胎停滞在球形向心形过渡期。在1-细胞期和2-细胞期时,野生型和突变体胚胎没有明显区别;野生型胚胎发育到球形期时,突变体胚胎主要处于8-16细胞期;野生型胚胎发育到心形期时,突变体胚胎主要处于早球形期;野生型胚胎继续发育为成熟胚胎,突变体胚胎大部分仍处于晚球形期,没有胚胎可以发育到心形期,突变体胚胎最终败育。突变体的胚胎除了发育迟缓,另一个明显的特征是细胞分裂异常,分裂板的位置发生变化,最终产生了形态各异的不规则的球形期胚。此外,突变体的胚乳核数目与野生型相比显著降低。FEM蛋白包含两个LRR结构和两个F-box结构域。GUS染色结果显示,FEM在幼苗、莲座叶、种荚、花序均有表达。在幼苗的子叶和新生真叶表达强烈,特别是在叶脉上表达明显,根上只有少量表达。直到胚胎发育到成熟期,FEM在胚乳和胚胎中一直有表达,在胚乳中表达强烈,在胚胎中相对较弱。以上结果表明,FEM基因对于胚胎和胚乳的发育是非常重要的。本论文第二部分工作是关于鼠李糖合成酶基因AtRHM1的功能分析。在植物进行正常的生命活动时,细胞壁是植物细胞不可缺少的重要部分。细胞壁不但在维持细胞形态与细胞之间黏结、决定细胞壁的强度和调控细胞伸长等方面起了必要的作用,而且还参与了细胞的分化、抗病、细胞识别与信号传导等一系列生理过程。细胞壁成分包含纤维素、半纤维素、果胶和少量的结构蛋白等。L-鼠李糖是拟南芥细胞壁果胶多聚糖的重要组分之一,在模式植物拟南芥中由三种鼠李糖合成酶AtRHM1,AtRHM2和AtRHM3催化合成。拟南芥中的RHM2基因的等位基因是MUM4(MUCILAGE-MODIFIED4)。RHM2基因突变导致拟南芥种皮粘液中鼠李糖和半乳糖醛酸含量的减少,进一步证实了RHM蛋白具有鼠李糖合成酶的功能。已经发现,RHM1参与拟南芥根毛的形成,但是AtRHM1蛋白的体内酶学活性和亚细胞定位仍然不清楚。本论文对AtRHM1的功能进行了进一步的阐述。AtRHM1与拟南芥中RHM家族的其它成员有很高的相似性,与其它植物例如水稻和葡萄也有高的相似性。AtRHM1启动子-GUS (Prhm1)融合表达结果表明AtRHM1几乎在各个组织器官表达,尤其在幼苗的根、子叶和植株花序表达强烈,GUS染色结果与RT-PCR结果一致。这与AtRHM3基因的表达模式相似,但AtRHM2基因的表达模式不同,AtRHM2/MUM4在繁殖器官中表达量较高,如种子等。对AtRHM1不同缺失启动子(Pd1,Pd2,Pd3,Pd4,Pd5)GUS转基因植株GUS染色结果表明:Prhm1和Pd1在整体上的GUS表达模式一致,即在整株幼苗都有表达,只是在染色程度上Pd1比Prhm1稍弱;而对于Pd2与Pd1染色情况比较来看,Pd2的子叶染色弱而根上几乎无染色,而Pd1的根上有很深的染色,表明RHM1启动子中的-752 bp～-308 bp这段序列可能与根特异表达相关;Pd3幼苗子叶上的染色较Pd2明显降低,2天时子叶无染色,4、7天的子叶只有顶尖处有稍许表达,表明-308 bp～-140 bp序列可能与子叶特异表达相关,而在RHM1基因启动子-752 bp～-308 bp含有两个G-box和一个与伤害有关的调控元件,在-308 bp～-140 bp区域中存在ACTT和ABRE元件。AtRHM1的表达受糖和伤害诱导,RHM1基因启动子中与伤害诱导相关的顺式作用元件可能位于-308 bp～140 bp,与葡萄糖应答相关顺式作用元件可能位于该基因启动子-931 bp～-752 bp区域。已有假设指出许多核糖转化酶定位在细胞质。Reiter和Pai指出烟草中一种UDP-D-apiose/UDP-D-xylose合成酶NbAXS1通常定位在细胞质。共聚焦显微镜观察表明GFP::AtRHM1融合蛋白定位在子叶细胞的细胞质,这说明AtRHM1是一种细胞质定位的蛋白。AtRHM1代表了RHM家族中第一个定位在细胞质的鼠李糖合成蛋白,这与许多其它参与细胞壁生物合成的核糖转化酶定位在细胞质相符。拟南芥中AtRHM1基因过表达导致叶片细胞壁的鼠李糖含量增加了40%,同时,甘露糖和葡萄糖的含量也发生了改变。而RNAi株系细胞壁中主要单糖包括鼠李糖的含量没有任何明显的改变。傅里叶变换红外光谱分析表明AtRHM1过表达株系中增加的鼠李糖参与了鼠李糖苷的合成。更多还原

【Abstract】 The thesis is constituted of two studies. The first is about an Arabidopsis F-box protein involved in embryogenesis. Precise control of gene expression is critical for embryo and endosperm development in plants. Recent studies revealed that the mutations of over 250 genes resulted in embryoic abnormalities or embryo lethalities, indicating these genes are essential for normal embryo development. The known embryo-defective genes encode proteins with diverse functions, including metabolism, cell growth, transcription, translation, protein fate, protein transport and traffic, and plant defense. In Arabidopsis, there’re more than 700 F-box proteins interacting with the rest of the SCF complex for degradation of the substrates. A number of studies have demonstrated that F-box proteins play critical roles in various signaling pathways, while some reports show that F-box proteins are also involved in embryo and endosperm development. The F-box proteins TIR1 and AFB were reported to mediate auxin responses during embryogenesis, and it’s found that the embryos of the triple mutant tir1-1 afb2-1 afb3-1 displayed defects during early embryogenesis. Other F-box proteins such as AtSFL61 and AtSFL70 are likely required for the development of embryo or female gametophyte. AtSFL61 mutation led to embryo-lethality with the frequent irregular cell division in atsfl61 embryos. Around 50% of the atsfl70/+ ovules could not survive beyond the zygotic division due to female gametophyte being defective. In this study, we report that F-BOX EMBRYO LETHAL (FEM), an F-box protein, is involved in embryogenesis and endosperm development in Arabidopsis. Loss-of-function mutations of FEM caused recessive embryo-lethal phenotype. Selfed heterozygous plants produced approximately 25% abnormal seeds which were shrunken and aborted. Most of the mutant embryos were arrested at globular stage and some at globular-to-heart transition stage. At 1-cell and 2-cell stages, it was difficult to distinguish the mutant from wild type; when wild-type ovules beared globular embryos, the mutant embryos were at 8-16-cell stage; when wild-type ovules contained heart-shaped embryos, the mutant embryos were at preglobular stage; when wild-type embryos developed mature embryos, the mutant embryos were arrested at globular-like or globular-to-heart transition stages and aborted. None of the fem embryos reached the heart stage. In addition to the embryos being arrested, aberrant cell divisions were observed in the mutant embryos with aberrant positioning of the division planes displayed, which resulted in globular embryos with irregular shape. Moreover, the number of endosperm nuclei of the mutant showed a dramatic reduction compared to wild type. FEM encodes a polypeptide with two LRR domains and two F-Box domains. FEM was ubiquitously expressed in various organs of transgenic Arabidopsis as revealed by FEM promoter-GUS fusion with a consistency with the microarray data (indicate the website). Detailed GUS staining with the transgenic ovules revealed the expression of FEM in the endosperm and developing embryos. Taken together, the data suggest that FEM gene is essential for both normal embryo and endosperm development in Arabidopsis.Second study is about the functional analysis of the Arabidopsis rhamnose biosynthesis gene AtRHM1. Cell wall plays essential roles during plant growth and development. The cell walls determine the boundaries of plant form, represent the main sink for photoassimilated carbon, protect against pathogen invasion, and provide mechanical strength to the plant body. Cell walls comprise primarily cellulose microfibrils, hemicelluloses, pectic polysaccharides, and small amounts of structural proteins. L-Rhamnose (Rha) is an important constituent of pectic polysaccharides, a major component of the cell walls of Arabidopsis, which is synthesized by three rhamnose biosynthesis enzymes (RHM) encoded by AtRHM1, AtRHM2, and AtRHM3, respectively. Mutations in Arabidopsis RHM2 gene, which are allelic to the MUCILAGE-MODIFIED4 (MUM4) locus, reduced the Rha and galacturonic acid content of seed coat mucilage, suggesting that RHM2/MUM4 is involved in RGI biosynthesis. Despite the finding that RHM1 is involved in root hair formation in Arabidopsis, experimental evidence is still lacking for the in vivo enzymatic activity and subcellular compartmentation of the AtRHM1 protein. AtRHM1 displays high similarity to the other members of RHM family in Arabidopsis and in other plant species such as rice and grape. Expression studies with AtRHM1 promoter-GUS (Prhm1) fusion gene showed that AtRHM1 was expressed almost ubiquitously, with more expression in roots and cotyledons of young seedlings and inflorescences, which is consistent with the results of RT-PCR. We made a series of AtRHM1 promoter deletion (Pd1,Pd2,Pd3,Pd4,Pd5). These deletion fragments were fused with GUS reporter gene respectively. The GUS staining showed that the expression pattern of Prhm1 and Pd1 was almost the same, whereas expression levels were different. For Pd1, expression level was a little bit reduced. Compared the expression level of Pd2 to Pd1, no staining was in cotyledon for Pd2, but strong staining was found for Pd1, indicating that -752 bp～-308 bp of RHM1 promoter was related to special expression of root. For Pd3, expression level in cotyledon of young seedling was reduced dramatically compared to Pd2. No staining was in 2 d seedling and a little staining was on top of 4 d and 7 d seedlings for Pd3, indicating that -308 bp～-140 bp was possibly involved in special expression of cotyledon. There were two G-box and a regulatory element of wounding in a range from -752 bp to -308 bp and ACTT and ABRE elements in a range from -308 bp～-140 bp. GUS staining showed that the expression of RHM1 was induced by sugar and wounding. -308 bp～140 bp of RHM1 promoter was possibly a regulatory element of wounding and -931 bp～-752 bp of RHM1 promoter was possibly involved in glucose answering reaction. It has been proposed that many nucleotide sugar interconverting enzymes are localized in cytosol. Reiter and Pai demonstrated that tobacco NbAXS1, a UDP-D-apiose/UDP-D-xylose synthase was mostly localized in the cytosol. GFP::AtRHM1 fusion protein was found to be localized in the cytosol of cotyledon cells, indicating AtRHM1 is a cytosol-localized protein. The overexpression of AtRHM1 gene in Arabidopsis resulted in an increase of rhamnose content as much as 40% in the leaf cell wall compared to the wild type, as well as an alteration in the contents of galactose and glucose. We did not find any significant difference (P < 0.05) in the amounts of the major sugars of cell wall material between wild type and RNAi line including Rha. Fourier-Transform Infrared analyses revealed that surplus rhamnose upon AtRHM1 overexpression contributes to the construction of rhamnogalacturonan.更多还原