【作者】 胡俊；

【导师】 孙蒙祥；

【作者基本信息】 武汉大学 ， 发育生物学， 2011， 博士

【摘要】 花粉外壁是包裹在花粉粒最外侧的一层物理化学性质非常稳定的特化细胞壁。花粉外壁的首要作用是保护花粉粒不受外界因素的伤害,防止花粉粒因失水而丧失活力,同时也为含有精细胞的花粉营养细胞质提供物理支持和保护,维持细胞膜的完整性以使花粉能持续正常发育。此外,花粉外壁的某些蛋白还在花粉与柱头的相互识别中起重要作用。因此,花粉外壁在花粉发育和受精过程中起着重要作用。在本实验室以前的研究中,通过差减抑制杂交技术筛选出了一些在烟草卵细胞和合子中差异表达的基因,并通过RT-PCR确定了一个在烟草花药和合子中特异表达的NtZC8基因,生物信息学分析表明NtZC8可能编码一个在花药和合子发育过程中起作用的甲基化转移酶蛋白。为了研究甲基化转移酶在花粉和合子发育过程中所起的调控作用,我们通过NCBI Blast搜索得到了6个拟南芥中与NtZC8基因同源性较高的基因,并对它们的表达模式进行了初步的研究。根据这6个基因在拟南芥花序和角果中表达水平的变化,我们选择其中一个基因,将其命名为EFD,并对其进行了详细研究。主要研究结果简述如下：EFD基因编码一个由323个氨基酸组成的蛋白,生物信息学分析表明其具有典型的甲基化转移酶结构域。对EFD蛋白三维结构进行结构建模发现其具有保守的甲基化转移酶结构形式,分析EFD蛋白序列则发现其含有以DNA分子为底物的DNA甲基化转移酶特有的结构域。将EFD蛋白与植物和动物中已知的几种类型的DNA甲基化转移酶进行进化关系分析,结果表明EFD蛋白与具有denovo甲基化活性的Dnmt3家族的甲基化转移酶的亲缘关系最近。为了观察EFD蛋白在细胞中的定位,我们构建了EFD蛋白C端融合sGFP蛋白的表达载体,基因枪转化洋葱表皮细胞发现融合蛋白特异定位于细胞核中。EFD蛋白在细胞核中的特异定位与其DNA甲基化转移酶的蛋白性质相吻合。为了更仔细的观察EFD基因的时空表达模式,我们构建了EFD启动子驱动GUS报告基因的表达载体,转化野生型拟南芥植株后观察转基因植株中的GUS信号。观察结果表明,EFD在花序中的表达呈现出时空特异性,它在发育早期的花药和雌蕊中表达水平较高。在小孢子发育过程中,EFD基因在早期的绒毡层细胞、小孢子母细胞和小孢子中均有表达,在单核小孢子晚期之后则只在绒毡层细胞中表达。对GUS信号的观察表明EFD基因可能参与了小孢子和雌蕊的早期发育。为了研究EFD基因的功能,我们以edf突变体为材料,观察EFD基因缺失所导致的异常表型。我们发现edf的育性受到了严重影响,基因功能补偿实验表明此不育表型确实是由EFD基因功能缺失引起的。正反交实验结果表明,edf突变体的雄性生殖系统的发育受到了严重影响。分析efd突变体的角果发现63%位于主苔的角果和75%位于侧苔的角果内完全没有形成种子,仅15.17%位于主苔的角果和7.92%位于侧苔的角果能发育成与野生型相似的角果。按照角果发育的顺序对角果进行观察,发现efd早期形成的角果几乎完全败育,仅末期可以形成少数育性正常的角果。对efd突变体各时期的小孢子和花粉的细胞核进行染色观察,发现efd花粉的减数分裂和有丝分裂进程正常,能够产生三核花粉,表明efd小孢子发生和花粉发育过程中细胞核的分裂无异常。对各发育时期的efd花药进行切片观察,发现四分体时期包裹着四分体的胼胝质壁较薄；efd单核小孢子呈现出胞质凝集并在随后出现空泡状；成熟花粉时期的efd花药内有类似于绒毡层降解物的残余物质游离在药室内或是附着于花粉表面。这一结果表明efd小孢子发育的异常可以追溯到四分体时期。从花药中分离各时期的小孢子进行观察,发现efd四分体的形态已出现异常,其胼胝质壁显得单薄粗糙,四个小孢子之问的分隔不明显,细胞干瘪,显得缺少细胞质内容物；efd单核小孢子的形态也与野生型不同,细胞表面粗糙不平；二核和三核时期的efd花粉中有一些发育停滞在单核阶段的小孢子和形态异常的花粉粒。这一观察结果表明虽然从四分体时期开始,efd花粉的发育已经出现异常,部分小孢子在发育过程中停滞死亡,但是还是有一些小孢子发育成为了三核成熟花粉。efd四分体时期胼胝质壁的形成异常,可能影响花粉外壁的正常发育。对成熟的efd花粉进行活力检测,发现约75.4%的成熟efd花粉活力良好,但是只有22.86%的efd花粉能够在体外萌发产生花粉管。对efd授粉后2天的角果进行染色观察,发现efd角果内没有任何花粉管形成。这一结果表明尽管75.4%的成熟efd花粉具有良好的活力,但是这些花粉在体内和体外萌发的能力都受到了严重影响。对efd花药进行环境扫描观察,发现花药表面的花粉粒的形状不规则,花粉之间还有类似于绒毡层降解片段的物质附着缠绕。观察花粉表面结构发现,efd花粉表面没有正常的立体纹饰结构,有的花粉表面光滑,有的花粉表面只有简单的小圆锥形凸起状结构。这一结果表明,虽然efd能够形成三核花粉,但是花粉的形态和表面纹饰结构都与野生型花粉明显不同,表明efd花粉的外壁发育受到了严重影响。对各时期efd花粉进行超薄切片观察其外壁发育,发现efd四分体内几乎没有原外壁形成,从四分体中释放出来的小孢子和随后形成的二核、三核花粉都没有外壁外层结构,在三核花粉阶段绒毡层降解物游离于药室内或是附着于花粉表面。这一观察结果表明efd原外壁的发育异常可能导致形成外壁的物质因缺乏模板而无法定向沉积,这可能是花粉外壁发育缺陷的主要原因。对efd花序转录组水平的变化进行研究,发现其中表达水平变化明显的基因都是与花药和花粉发育相关的基因。这些基因中,有的与胼胝质合成相关的,有的与原外壁的正常发育相关,还有一些与孢粉素的合成、分泌和转运相关。这一结果表明,在efd突变体中,与花粉外壁发育相关的三大重要因素——胼胝质、原外壁和孢粉素都受到了影响,最终导致efd花粉外壁出现严重缺陷。综上所述,在本研究中我们使用生物信息学手段在拟南芥中找到了一个编码DNA甲基化转移酶、与花药早期发育相关的EFD基因。对此基因的突变体所做的研究表明,EFD基因参与了小孢子发育早期胼胝质和原外壁形成的调控,证实了此基因在拟南芥花粉外壁的发育过程中具有十分重要的调控作用,为今后进一步研究小孢子发育和甲基化调控之间的关系提供了新的启示。更多还原

【Abstract】 Exine, a chemically and physically resistant structure, is the outermost layer of pollen wall. Exine protects pollen grains from harsh conditions and prevents water loss from pollen grains to keep them viable. Exine also provides physicl support to pollen vegetative cell containing sperm. Besides, proteins existed in exine are proved to be important for recognization and adherence between pollen and stigma. Thus, the exine plays very important roles in pollen development and fertilization.In previous study in our lab, we have found out several genes that specifically expressed in egg and zygoty in tobacco using suppression subtractive hybridization, and identified one of those genes, NtZC8, that specifically expressed in anther and zygote. Bioinformatics analysis indicates that NtZC8is likely to encode a methyltransferase that is invoved in pollen and zygote development.To seek the role of methyltransferase in pollen and zygote development we found six genes in Arabidopsis, which are high homology to NtZC8. Accordinding to their expression pattern revealed by RT-PCR, we then chose one of these genes, named EFD, for further study. The main results are as follows:EFD encodes a323amino acid protein with a methyltransferase domain, structural analysis showed that EFD is conserved in a typical methyltransferase structure and a signature motif of DNA methytransferase is also found in EFD. Evolutionary analysis also indicates that EFD has the closest relationship with proteins of Dnmt3family, which usually function in de novo methylation.Expression vector with EFD fused with sGFP driven by EFD native promoter is constructed and bombarded into onion epidermal cells to detect subcellular location of EFD protein, the result shows that EFD fusion protein is only detected in the nucleus, which is consistent to the nuclear location of DNA methytransferases.Spatial and temporal patterns of EFD expression during anther development are determined by GUS reporter gene. The results show that EFD is strongly expressed in early-staged buds of inflorescence, in both stamens and pistils; sections of stained buds revealed that EFD is expressed in tapetum and microsporocyte, tetrad and early-staged microspore. Later, it is expressed only in tapetum when mitosis begins. The GUS report gene reveals that EFD may be involved in early development in microspores and pistils.To study the functions of EFD, we get a knock-out mutant of EFD and observe whether there are abnormalities in this mutant. We first find that fertility of efd is severely affected, complementation of EFD can rescue this sterility and reciprocal corsses show that male reproductive system is impaired. These results indicate that loss of EFD fuction leads to male steritlity.Ananlysis of efd siliques reveals that63.76%efd siliques on main stalk and75.89%on lateral stalk are totally sterility and only15.17%efd siliques on main stalk and7.92%on lateral stalk look like normal wild type siliques. We also find that aborted siliques are produced in the early stage of florescence and siliques with viable seeds are only produced at the late stage of florescence.We staied nucleus of microspores and pollen grains and find that efd microspores can go through meiosis and mitosis to develop into trinucleate pollen, which indicates that nucleus division is normal in efd microsporogenesis and pollen development.We made anther cross sections to observe anther development in efd mutant. At stage7, the callose wall of efd tetrad looked thinner than that in wide type. At stage8when callose wall degenerated and microspores were released, microspores of efd looked condensed and the callose degeneration was not coinstantaneous in different loculus in the same anther; from stage9to11, some efd microspores looked empty or irregular in shape. At stage12, there were some tapetum residents in anther locule while some pollen grains looked like normal wild type pollen grains in efd mutant.We anatomized anthers of both wild type and efd and compared them with each other. At the late tetrad stage, tetrads from efd were surrounded by the callose layer which was not as thick as that of wild type, and cells in tetrad were not well isolated by the callose wall. When uni-nucleus microspores released from tetrads, efd microspore surface looked coarse. When the mitosis began, some efd microspores could go through such process and developed into mature pollen grains while some stoped at uni-nuclei microspore stage and some collapse. These results indicate that microsporogenesis of efd is aberrant since tetrad pollen stage and callose wall formation is affected in efd, which may lead to exine defects.FDA stating showed that75%mature efd pollen was viable. But both in vivo and in vitro pollen germination were severaly impaired in efd, no pollen tube could be observed in siliques2days after fertilization, and only22.86%efd pollen grains could germinate in vitro. These results indicate efd pollen development is obviously affected so that mature pollen can hardly germinate to carry out double fertilization although most efd pollens are alive.Environmental scanning electron microscopy showed that efd pollen grains were aborted and irregular in shape, the surface of mutant pollen grains were smooth, some membrane-like fragments, which looked like tapetum remnants, were adherent to pollen grains. This resulte indicates that the exine formation is affected in efd mutant. Transmission electron microscopy confirmed that exine was almost totally missing in efd mutant. In efd, callsoe deposited to form a thin callose wall, but no obviouse primexine structure could be observed between callose layer and plasma membrane, no exine-like strcutre can be observed when microspores were released from tetrads; debris of tapetal cells could be found in efd anther, some of which attached to the surface of efd pollen grains and the others just remained in the anther locule. Thus, the failure of primxine formation is supposed to be an important factor resulting in exine development defects.We also investigated the transcriptome of edf and found that most chaning genes are involved in anther and pollen development. Some genes known to be involved in Arabidopsis callose wall formation, primexine formation, sporopollenin syntheisis or transfer are found down-regulated in efd mutant, which indicates that three key factors for exine formation, callose, primexine and sporopollenin are all affected in efd mutant and lead to failure of exine formation in efd.更多还原