拟南芥绒毡层和中层的基因调控网络

发布时间：2018-07-17 00:42

【摘要】：花药的主要功能是产生有功能的花粉以及成熟花粉的及时释放。模式植物拟南芥的花药四层体细胞壁包括表皮层、药室内壁、中层和绒毡层。其中,绒毡层作为花药壁的最内层,与花粉形成密切相关,为发育中的小孢子提供营养、胼胝质酶,以及花粉外壁物质等;而药室内壁与花粉的释放相关。近几年,克隆了多个绒毡层发育的关键基因,但是对绒毡层基因的精细调控网络仍不清楚。中层介于药室内壁和绒毡层之间,对其发育和基因表达了解非常有限。本文运用激光显微切割技术分别特异地收集拟南芥花药四分体时期前后的绒毡层细胞,抽提RNA,质检合格后反转录成cDNA,接着进行转录组测序。98%的Reads可以比对到拟南芥参考基因组,几乎全部富集在参考基因组的CDS元件上。早晚期的绒毡层细胞中分别有18298和18227个的基因表达。早期绒毡层细胞中显著表达的基因,主要参与RNA转录调控,蛋白翻译与修饰,各种酶类的合成以及细胞发育过程等;晚期绒毡层细胞中显著表达的基因,则主要参与蛋白降解,信号转导,糖类等的转运以及细胞壁的降解等。已知绒毡层发育的遗传调控通路DYT1-TDF1-AMS-MS188,将这四个转录因子突变体的芯片数据与绒毡层细胞的转录组数据进行综合分析。分析发现,dyt1突变体下有1548个基因在绒毡层中表达,dyt1,tdf1共同下调的基因1354个,dyt1,tdf1,ams共同下调的基因985个,有810个基因在dyt1,tdf1,ams, ms188中均下调。将在dyt1下调但不在tdf1下调的基因定义为DYT1特异调控的基因,以此类推,DYT1特异调控的基因有194个,TDF1、AMS和MS188特异调控的基因分别有369、175和810个。DYT1参与绒毡层的早期发育;:TDF1继续调节绒毡层的发育,并为绒毡层的PCD作准备;AMS启动花粉壁的形成和绒毡层的PCD; M188主要调控绒毡层细胞壁的降解和花粉壁的形成。激素可能参与到特定阶段的绒毡层发育。生长素是DYT1时期的主要激素,乙烯信号转导基因在TDF1阶段明显富集,生长素和脱落酸相关基因在MS188阶段富集。DYT1、TDF1、AMS这三个绒毡层发育早期的转录因子分别调节一些特定的E3泛素连接酶,从而通过泛素化蛋白降解途径去降解一些蛋白质。综上,DYT1-TDF1-AMS-MS188这条遗传通路以及各转录因子调节的下游基因共同形成了复杂的绒毡层基因调控网络。由于拟南芥中层细胞很难用激光显微切割技术分离,本文利用激光显微切割技术收集百合中层细胞并进行转录组测序。共获得了207 792 808条Reads,拼接后得到65029条Unigene。将所获得的Unigene与NR、SWISSPROT、KOG、GO和KEGG这5个公共数据库进行比对,结果发现,分别有27156, 21367, 17654, 8828,19784条Unigene可比对到以上数据库中。已注释的Unigene比对到NR数据库中的主要物种为水稻和玉米;与KOG数据库比对后按功能共分为24类,主要为一般性的功能预测,转录后调控,信号转导,细胞运输和分泌与囊泡运输;经过与KEGG数据库比对后按照代谢通路可分为211类,有46.68%与代谢本身相关,其余与遗传信息处理、环境信息交互处理、细胞进程相关;根据GO功能可分为3大类64小类,主要包含细胞和膜的细胞组分,具有结合和催化活性的分子功能,参与代谢、细胞和调控生物学过程。综合基因功能注释结果,拟南芥中层细胞中表达的基因有4763个,包括RPK2,EMS,BAM1,BAM2,SERK1, SERK2,ROXY1,TDF1这些与绒毡层分化和发育相关的基因。中层细胞的转录组信息为今后进行中层细胞特异的分子标记的开发和关键基因的克隆及功能分析等研究提供基础数据。此外,本文通过凝胶迁移阻滞实验体外证明AMS可以直接结合于bHLH089启动子上,但是染色质免疫共沉淀实验体内并不能证明AMS能结合到bHLH010,089,091的启动子上,同时烟草瞬时表达系统表明,bHLH010,089,091的启动子在烟草体系中能被启动,并不适用烟草瞬时表达实验。然而,Rescue实验获得ams变体背景的PoAMS: bHLH010, 089, 091转基因株系不能恢复各自的表达,也不能恢复ams的突变表型。这些结果表明,在绒毡层、小孢子和中层中均有表达的三个功能冗余的bHLH转录因子bHLH010,089,091与AMS之间并不是简单的上下游调控关系,可能存在蛋白互作和反馈调节。综上,绒毡层和中层与花粉的发育和成熟息息相关,存在许多基因形成的复杂精细的基因调控网络。
[Abstract]:The main function of anthers is the production of functional pollen and the timely release of mature pollen. The four layer body cell wall of the anther of Arabidopsis thaliana consists of the epidermis, the inner wall of the pharmacy, the middle layer and the tapetum. The tapetum is the most inner layer of the anther wall, closely related to the pollen formation, and provides nutrition, callose enzyme for the microspores in the developing. In recent years, the fine regulation network of the tapetum gene is still not clear. The middle layer is between the inner wall of the medicine chamber and the tapetum, and the development and the expression of the base are very limited. The tapping technique collects the tapetum cells of the four division of Arabidopsis anther in a special way, and extracts RNA, and then transcripted into cDNA after the quality examination is qualified. Then the Reads of the transcriptional sequence.98% can be compared to the Arabidopsis reference genome, almost all of the CDS elements in the reference genome are enriched. There are 18 of the tapetum cells in the early and late stage of the tapetum. The gene expression of 298 and 18227. The genes expressed in the early tapetum cells are mainly involved in RNA transcription regulation, protein translation and modification, the synthesis of various enzymes, and the process of cell development. The genes that are expressed in the late tapetum cells are mainly involved in the protein degradation, signal transduction, carbohydrate transport, and cell wall The genetic regulation pathway DYT1-TDF1-AMS-MS188, known as the tapetum development, is known as the integrated analysis of the transcriptional data of the tapetum cells and the chip data of these four transcription factor mutants. It was found that 1548 genes were expressed in the tapetum, and 1354 genes, DYT1, and tdf1, were co regulated by the DYT1 mutant, DYT1, tdf1, and AMS together. 985 genes were down regulated, and 810 genes were down regulated in DYT1, tdf1, AMS, and ms188. The genes that were down regulated in DYT1 but not down regulated by tdf1 were defined as DYT1 specific genes. By this analogy, 194 genes regulated by DYT1, 369175 and 810.DYT1 specifically regulated by TDF1, AMS and MS188, were involved in the early development of the tapetum, respectively. TDF1 continues to regulate the development of tapetum and prepare for the PCD of the tapetum; AMS starts the formation of the pollen wall and the PCD of the tapetum; M188 mainly regulates the degradation of the tapetum cell wall and the formation of the pollen wall. Hormones may be involved in the development of the tapetum at a specific stage. Auxin is the main hormone in the DYT1 period, and the ethylene signal transduction gene is in TDF1. At the stage of MS188, the genes of auxin and abscisic acid are enriched in the MS188 stage to enrich.DYT1, TDF1, and AMS, which regulate some specific E3 ubiquitin ligase, respectively, to degrade some proteins through the ubiquitin protein degradation pathway. To sum up, DYT1-TDF1-AMS-MS188 this genetic pathway and the various transcripts. A complex tapetum gene regulation network is formed by factor regulated downstream genes. Because the middle layer cells of Arabidopsis are difficult to be separated by laser microdissection, laser microdissection is used to collect middle layer cells of Lilium and to be sequenced in transcriptional groups. 207792808 Reads are obtained and 65029 Unigene. will be obtained after splicing. The obtained Unigene is compared with 5 public databases such as NR, SWISSPROT, KOG, GO and KEGG. The results show that 27156, 21367, 17654, 882819784 Unigene can be compared to the above database. The annotated Unigene ratio is the main species in the NR database for paddy rice and corn; compared with the KOG database, the function is divided into 24. Class, mainly for general function prediction, post transcriptional regulation, signal transduction, cell transport and secretion and vesicle transport; after comparison with the KEGG database, the metabolic pathways can be divided into 211 categories, 46.68% are related to metabolism itself, the rest is related to genetic information processing, environmental information interaction, and cell processes related; according to the function of GO, it can be divided into 3 major categories. Class 64 small classes, mainly including cell and membrane cell components, molecular functions with binding and catalytic activity, involved in metabolism, cell and biological processes. Comprehensive gene functional annotation results, 4763 genes expressed in Arabidopsis middle layer cells, including RPK2, EMS, BAM1, BAM2, SERK1, SERK2, ROXY1, TDF1, and tapetum differentiation and hair The related genes. The transcriptional information of the middle layer cells provides basic data for the development of specific molecular markers in the middle layer and the cloning and functional analysis of key genes. In addition, this paper has demonstrated that AMS can be directly combined with bHLH089 promoter in vitro by gel migration block test, but chromatin immuno sedimentation The AMS can not be proved to be combined with the promoter of bHLH010089091. Meanwhile, the transient tobacco expression system shows that the promoter of bHLH010089091 can be started in the tobacco system and does not apply to the transient expression of tobacco. However, the Rescue experiment obtained the PoAMS: bHLH010 of the AMS variant background, 089, 091 transgenic lines can not be restored. The results showed that the three functional redundant bHLH transcription factors, bHLH010089091 and AMS, between the tapetum, the microspore and the middle layer, were not a simple upstream and downstream regulation relationship between the tapetum, the microspore and the middle layer, and there might be protein interaction and feedback regulation. To sum up, the tapetum and the middle layer and the pollen could be found. The development and maturation are closely related, and there are many complex and fine gene regulatory networks formed by many genes.
【学位授予单位】：上海师范大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：Q943.2

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