擬南芥絨氈層和中層的基因調(diào)控網(wǎng)絡
發(fā)布時間：2018-07-17 00:42
【摘要】：花藥的主要功能是產(chǎn)生有功能的花粉以及成熟花粉的及時釋放。模式植物擬南芥的花藥四層體細胞壁包括表皮層、藥室內(nèi)壁、中層和絨氈層。其中,絨氈層作為花藥壁的最內(nèi)層,與花粉形成密切相關,為發(fā)育中的小孢子提供營養(yǎng)、胼胝質(zhì)酶,以及花粉外壁物質(zhì)等;而藥室內(nèi)壁與花粉的釋放相關。近幾年,克隆了多個絨氈層發(fā)育的關鍵基因,但是對絨氈層基因的精細調(diào)控網(wǎng)絡仍不清楚。中層介于藥室內(nèi)壁和絨氈層之間,對其發(fā)育和基因表達了解非常有限。本文運用激光顯微切割技術分別特異地收集擬南芥花藥四分體時期前后的絨氈層細胞,抽提RNA,質(zhì)檢合格后反轉錄成cDNA,接著進行轉錄組測序。98%的Reads可以比對到擬南芥參考基因組,幾乎全部富集在參考基因組的CDS元件上。早晚期的絨氈層細胞中分別有18298和18227個的基因表達。早期絨氈層細胞中顯著表達的基因,主要參與RNA轉錄調(diào)控,蛋白翻譯與修飾,各種酶類的合成以及細胞發(fā)育過程等;晚期絨氈層細胞中顯著表達的基因,則主要參與蛋白降解,信號轉導,糖類等的轉運以及細胞壁的降解等。已知絨氈層發(fā)育的遺傳調(diào)控通路DYT1-TDF1-AMS-MS188,將這四個轉錄因子突變體的芯片數(shù)據(jù)與絨氈層細胞的轉錄組數(shù)據(jù)進行綜合分析。分析發(fā)現(xiàn),dyt1突變體下有1548個基因在絨氈層中表達,dyt1,tdf1共同下調(diào)的基因1354個,dyt1,tdf1,ams共同下調(diào)的基因985個,有810個基因在dyt1,tdf1,ams, ms188中均下調(diào)。將在dyt1下調(diào)但不在tdf1下調(diào)的基因定義為DYT1特異調(diào)控的基因,以此類推,DYT1特異調(diào)控的基因有194個,TDF1、AMS和MS188特異調(diào)控的基因分別有369、175和810個。DYT1參與絨氈層的早期發(fā)育;:TDF1繼續(xù)調(diào)節(jié)絨氈層的發(fā)育,并為絨氈層的PCD作準備;AMS啟動花粉壁的形成和絨氈層的PCD; M188主要調(diào)控絨氈層細胞壁的降解和花粉壁的形成。激素可能參與到特定階段的絨氈層發(fā)育。生長素是DYT1時期的主要激素,乙烯信號轉導基因在TDF1階段明顯富集,生長素和脫落酸相關基因在MS188階段富集。DYT1、TDF1、AMS這三個絨氈層發(fā)育早期的轉錄因子分別調(diào)節(jié)一些特定的E3泛素連接酶,從而通過泛素化蛋白降解途徑去降解一些蛋白質(zhì)。綜上,DYT1-TDF1-AMS-MS188這條遺傳通路以及各轉錄因子調(diào)節(jié)的下游基因共同形成了復雜的絨氈層基因調(diào)控網(wǎng)絡。由于擬南芥中層細胞很難用激光顯微切割技術分離,本文利用激光顯微切割技術收集百合中層細胞并進行轉錄組測序。共獲得了207 792 808條Reads,拼接后得到65029條Unigene。將所獲得的Unigene與NR、SWISSPROT、KOG、GO和KEGG這5個公共數(shù)據(jù)庫進行比對,結果發(fā)現(xiàn),分別有27156, 21367, 17654, 8828,19784條Unigene可比對到以上數(shù)據(jù)庫中。已注釋的Unigene比對到NR數(shù)據(jù)庫中的主要物種為水稻和玉米;與KOG數(shù)據(jù)庫比對后按功能共分為24類,主要為一般性的功能預測,轉錄后調(diào)控,信號轉導,細胞運輸和分泌與囊泡運輸;經(jīng)過與KEGG數(shù)據(jù)庫比對后按照代謝通路可分為211類,有46.68%與代謝本身相關,其余與遺傳信息處理、環(huán)境信息交互處理、細胞進程相關;根據(jù)GO功能可分為3大類64小類,主要包含細胞和膜的細胞組分,具有結合和催化活性的分子功能,參與代謝、細胞和調(diào)控生物學過程。綜合基因功能注釋結果,擬南芥中層細胞中表達的基因有4763個,包括RPK2,EMS,BAM1,BAM2,SERK1, SERK2,ROXY1,TDF1這些與絨氈層分化和發(fā)育相關的基因。中層細胞的轉錄組信息為今后進行中層細胞特異的分子標記的開發(fā)和關鍵基因的克隆及功能分析等研究提供基礎數(shù)據(jù)。此外,本文通過凝膠遷移阻滯實驗體外證明AMS可以直接結合于bHLH089啟動子上,但是染色質(zhì)免疫共沉淀實驗體內(nèi)并不能證明AMS能結合到bHLH010,089,091的啟動子上,同時煙草瞬時表達系統(tǒng)表明,bHLH010,089,091的啟動子在煙草體系中能被啟動,并不適用煙草瞬時表達實驗。然而,Rescue實驗獲得ams變體背景的PoAMS: bHLH010, 089, 091轉基因株系不能恢復各自的表達,也不能恢復ams的突變表型。這些結果表明,在絨氈層、小孢子和中層中均有表達的三個功能冗余的bHLH轉錄因子bHLH010,089,091與AMS之間并不是簡單的上下游調(diào)控關系,可能存在蛋白互作和反饋調(diào)節(jié)。綜上,絨氈層和中層與花粉的發(fā)育和成熟息息相關,存在許多基因形成的復雜精細的基因調(diào)控網(wǎng)絡。
[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.
【學位授予單位】：上海師范大學
【學位級別】：博士
【學位授予年份】：2017
【分類號】：Q943.2
[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.
【學位授予單位】：上海師范大學
【學位級別】：博士
【學位授予年份】：2017
【分類號】：Q943.2
【參考文獻】
相關期刊論文 前5條
1 ;A Genetic Pathway for Tapetum Development and Function in Arabidopsis[J];Journal of Integrative Plant Biology;2011年11期
2 ;Gibberellin and Jasmonate Crosstalk during Stamen Development[J];Journal of Integrative Plant Biology;2009年12期
3 宋W，
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