本文選題：雌配子體 + 胚囊��； 參考：《浙江大學(xué)》2016年博士論文

【摘要】：被子植物具有雌、雄兩種配子體,其生活周期是二倍體孢子體和單倍體配子體世代交替的過程,其中,雌配子體(胚囊)在植物花粉管引導(dǎo)、受精和受精后的種子發(fā)育等生殖過程扮演著關(guān)鍵角色。雌配子體的發(fā)育是非常重要的發(fā)育事件之一,在基礎(chǔ)理論研究和農(nóng)業(yè)育種工作中具有重要意義,一直是植物生殖發(fā)育生物學(xué)研究的熱點。研究表明,雙組分信號系統(tǒng)(Two-component system, TCS)介導(dǎo)的磷酸化傳遞是植物調(diào)節(jié)細(xì)胞內(nèi)信號轉(zhuǎn)導(dǎo)的主要機(jī)制之一。被子植物具有包括組氨酸激酶(Histidine kinase, HK)、組氨酸磷酸轉(zhuǎn)移蛋白(Histidine phosphotransferprotein,HP)和反應(yīng)調(diào)節(jié)因子(Response regulator, RR)的多步磷酸化傳遞的雙組分信號系統(tǒng)。目前對植物雙組分信號系統(tǒng)功能的研究主要集中在逆境脅迫和營養(yǎng)生長方面,對生殖發(fā)育尤其是TCS對雌配子體發(fā)育和細(xì)胞命運分化的作用還知之甚少。為進(jìn)一步揭示植物雙組分信號系統(tǒng)與雌配子體發(fā)育和細(xì)胞命運決定的關(guān)系,本研究對模式植物擬南芥胚囊發(fā)育過程中的雙組分信號進(jìn)行了檢測,并分析了參與雙組分信號轉(zhuǎn)導(dǎo)的組氨酸激酶基因在胚囊中的表達(dá)模式,重點研究了細(xì)胞分裂素受體對功能大孢子形成的調(diào)控以及CKI1 (cytokinin-indepenndent 1)基因與雌配子體細(xì)胞命運決定的關(guān)系。同時,為深入研究白菜中的雙組分信號系統(tǒng),基于白菜全基因組對白菜IPTs (isopentenyl transferases)和(CKXs (cytokinin oxidase/dehydrogenases)基因家族、TCS基因以及CRFs (cytokinin response factors)基因家族進(jìn)行了鑒定,并對上述基因家族的基因組信息、蛋白特征、進(jìn)化和表達(dá)模式等方面進(jìn)行了研究,為今后開展其功能的分析奠定了基礎(chǔ)。主要研究結(jié)果如下：(1)利用TCSnpro::NLS-3XeGFP marker對擬南芥胚囊發(fā)育過程中雙組分信號的動態(tài)分布進(jìn)行了觀察,結(jié)果表明,雙組分信號從胚囊發(fā)育的功能大孢子時期到成熟期在雌配子細(xì)胞中均存在,暗示雙組分信號系統(tǒng)在雌配子體發(fā)育過程中具有重要的功能。對所有能夠參與雙組分信號轉(zhuǎn)導(dǎo)的7個組氨酸激酶基因在胚囊發(fā)育過程中啟動子活性的分析結(jié)果表明,除AHK2外,在胚囊中AHK1、 AHK3、AHK4、AHK5、CKI1和ETRl均檢測到表達(dá)信號,且CKI1表達(dá)相對較高。對ahk2 ahk3 ahk4三突變體的觀察結(jié)果表明,AHK2,AHK3和AHK4在調(diào)控功能大孢子發(fā)育時存在功能冗余。(2)擬南芥ckil-9/+突變體表現(xiàn)出雌配子體的敗育表型。對胚珠DIC觀察表明,突變體胚囊中兩個極核往往不能正常融合,還伴隨著未發(fā)生降解退化的三個反足細(xì)胞核從胚囊合點端向珠孔端的遷移。構(gòu)建了雌配子體細(xì)胞特異表達(dá)的單1narker和雙marker,并將上述marker通過擬南芥浸花轉(zhuǎn)化法轉(zhuǎn)入ckil-9,/+突變體中,結(jié)果表明,突變體胚囊中反足細(xì)胞和中央細(xì)胞的細(xì)胞命運徹底丟失,三個反足細(xì)胞和兩個未融合的極核表現(xiàn)為卵細(xì)胞或者助細(xì)胞的細(xì)胞命運。對CKI1蛋白在胚囊發(fā)育過程中的定位分析表明,CKIl從胚囊發(fā)育的FG4時期開始表現(xiàn)出合點端的極性定位,這種極性定位可能是通過基因轉(zhuǎn)錄后的調(diào)節(jié)機(jī)制實現(xiàn)的。利用ES1啟動子在胚囊中過表達(dá)CKIl基因,結(jié)果顯示,CKI1在卵細(xì)胞和助細(xì)胞中的異位表達(dá)能夠?qū)⒙鸭?xì)胞和助細(xì)胞的細(xì)胞命運轉(zhuǎn)變成中央細(xì)胞的細(xì)胞命運�；谏鲜鼋Y(jié)果提出了雌配子體細(xì)胞命運決定的兩種模型。(3)對,IHPI～AHP5在成熟胚囊中啟動子活性的分析結(jié)果表明,AHP1基因在中央細(xì)胞和助細(xì)胞中表達(dá),AHP3基因在中央細(xì)胞中特異表達(dá),AHP2和AHP5基因在中央細(xì)胞、卵細(xì)胞、助細(xì)胞和反足細(xì)胞中均有表達(dá),而AHP4基因在胚囊中不表達(dá)。隨后構(gòu)建了ahp多突變體并對多突變體的種子發(fā)育和胚囊發(fā)育進(jìn)行了研究,結(jié)果顯示,ahp2-2 ahp3 ahp5-2/+三突變體表現(xiàn)出與ckil-9/+相似的表型,AHP2、AHP3和AHP5共同作用于CKIl基因的下游調(diào)控雌配子體的發(fā)育。(4)對植物HK家族蛋白氨基酸序列的比對發(fā)現(xiàn),CKI1在HK結(jié)構(gòu)域和HATPase結(jié)構(gòu)域中分別存在著氨基酸序列為SHD和GLGLG的特異motif,可以作為判定CKI1的重要依據(jù)。通過overlap extension PCR技術(shù)對CKI1結(jié)構(gòu)域的替換研究了CKI1的結(jié)構(gòu)域與CKI1基因功能的關(guān)系,結(jié)果表明,CKI1的HK結(jié)構(gòu)域和HATPase結(jié)構(gòu)域而非Rec結(jié)構(gòu)域影響CKI1基因的功能。另外,CKI1蛋白亞細(xì)胞定位的研究結(jié)果顯示,CKI1的N-端區(qū)域決定著CKI1蛋白在內(nèi)質(zhì)網(wǎng)上的定位。(5)對不同長度的CKI1啟動子的研究表明,CKIl的核心啟動子元件位于第一個內(nèi)含子中。利用酵母單雜技術(shù)對CKI1上游轉(zhuǎn)錄調(diào)控因子進(jìn)行了篩選和鑒定,最終篩選獲得了7個在中央細(xì)胞中表達(dá)的轉(zhuǎn)錄因子基因。(6)在白菜基因組中分別鑒定到了13個IPTs基因和12個CKXs基因,并對其基因組信息和蛋白特征做了分析。多序列比對、保守結(jié)構(gòu)域和系統(tǒng)進(jìn)化分析結(jié)果表明,白菜IPTs和CKXs都可以分為Ⅰ、Ⅱ、Ⅲ和IV四個亞組。選取白菜、擬南芥和琴葉擬南芥三個十字花科植物中的IPTs和CKXs做了基因組層面的比較分析,發(fā)現(xiàn)其基因存在片段重復(fù)和串聯(lián)重復(fù)現(xiàn)象,并通過計算直系同源基因和旁系同源基因的Ks和Ka值對基因的進(jìn)化模式做了分析。結(jié)果顯示,白菜中旁系同源基因的Ks值明顯比白菜、擬南芥和琴葉擬南芥中直系同源基因的Ks值要小,同時,白菜在大約26～33MYA與擬南芥分離開。另外,通過qRT-PCR技術(shù)對白菜IPTs和CKXs在不同器官中表達(dá)模式的分析,篩選出了一些器官特異表達(dá)的基因。對白菜IPTs和CKXs在干旱脅迫和鹽脅迫條件下以及對外源細(xì)胞分裂素和脫落酸響應(yīng)模式開展研究,獲得了一些參與非生物脅迫和激素調(diào)控網(wǎng)絡(luò)的候選基因。(7)對白菜中的雙組分信號元件進(jìn)行了鑒定,共找到了85個TCS基因成員,包括20個HK基因、8個HPs和57個RRs,并對其基因組信息和蛋白特征做了分析。多序列比對、保守結(jié)構(gòu)域和系統(tǒng)進(jìn)化分析結(jié)果表明,HKs可以分為細(xì)胞分裂素受體亞家族、AHKl亞家族、AHK5亞家族、CKI1亞家族、乙烯受體亞家族和光敏色素亞家族。RRs可以分為Type-A RR、Type-B RR、Type-C RR和pseudo-RR四大類。我們分析和比較了白菜和擬南芥中TCS基因的片段重復(fù)和串聯(lián)重復(fù)現(xiàn)象,并通過計算直系同源基因和旁系同源基因的Ks和Ka值對基因的進(jìn)化模式做了分析。另外,通過qRT-PCR技術(shù)對白菜TCSs在不同器官中的表達(dá)模式做了分析,篩選獲得了一些器官特異表達(dá)的基因。此外,還研究了白菜TCSs對干旱脅迫和鹽脅迫的響應(yīng)特征,以及對外源細(xì)胞分裂素、生長素和脫落酸的響應(yīng)特征。(8)在白菜基因組中鑒定出了281個AP2/ERF超級基因家族成員,并對其保守結(jié)構(gòu)域和進(jìn)化樹進(jìn)行了分析,結(jié)果表明,白菜AP2/ERF超級家族可以分為AP2家族、RAV家族和ERF家族,其中ERF家族又可以進(jìn)一步分為IREB亞家族和ERF亞家族,包括Ⅰ～Ⅺ 13個小組。對小組Ⅵ和VI-L中的21個CRFs做了重點研究。白菜CRFs可以分為Ⅰ～Ⅴ5個分支,編碼A、B和C三種類型的蛋白。利用qRT-PCR技術(shù)對白菜CRFs在不同器官中表達(dá)模式開展研究,結(jié)果表明,CRFs在不同器官中是遍在表達(dá)的,但不同部位的表達(dá)量存在一定差異。通過研究CRFs對干旱脅迫和鹽脅迫的響應(yīng)模式以及對外源細(xì)胞分裂素和脫落酸的響應(yīng)模式,篩選獲得了一批參與非生物脅迫和激素調(diào)控網(wǎng)絡(luò)的候選基因。研究結(jié)果能使我們從一個新的視角理解植物的雌配子體的發(fā)育過程,同時也為充分理解經(jīng)濟(jì)作物有性生殖的分子機(jī)制,完善蕓薹屬蔬菜作物的生殖生物學(xué)研究基礎(chǔ),有效調(diào)控作物育性,實現(xiàn)優(yōu)質(zhì)高產(chǎn)、高效繁殖提供新的理論依據(jù)和技術(shù)支持。
[Abstract]:The angiosperms have two gametophytes of female and male, whose life cycle is the process of alternation of diploid sporophyte and haploid gametophyte, in which the female gametophyte (embryo sac) plays a key role in the reproductive process, such as the pollen tube, fertilization and fertilization, and the development of the female gametophyte is one of the most important developmental events. The research shows that Two-component system (TCS) mediated phosphorylation is one of the main mechanisms for regulating intracellular signal transduction in plants. The angiosperms include histidine kinase, which includes histidine kinase. (Histidine kinase, HK), histidine phosphate transfer protein (Histidine phosphotransferprotein, HP) and reactive modulating factor (Response regulator, RR) multistep phosphorylation signaling system. The current research on the function of plant dual component signal system is focused on stress and nutritional growth, especially for reproductive development. The effect of TCS on the development of female gametophyte and cell fate differentiation is little known. In order to further reveal the relationship between the plant dual component signal system and the development of female gametophyte and cell fate, this study examined the two component signals in the development of the embryo sac of the model plant Arabidopsis thaliana, and analyzed the involvement of the two component signals. The expression pattern of the histidine kinase gene in the embryo sac focuses on the regulation of the cytokinin receptor on the formation of functional megaspore and the relationship between the CKI1 (cytokinin-indepenndent 1) gene and the fate determination of the female gametophyte. IPTs (isopentenyl transferases) and (CKXs (cytokinin oxidase/dehydrogenases) gene family, TCS gene and CRFs (cytokinin response factors) gene family were identified, and the genome information, protein characteristics, evolution and expression patterns of the above gene family were studied in order to carry out their functional analysis in the future. The main research results are as follows: (1) the dynamic distribution of two component signals during the development of the embryo sac of Arabidopsis thaliana was observed by TCSnpro:: NLS-3XeGFP marker. The results showed that the two component signals existed from the functional megaspore to the mature stage of the embryo sac, suggesting the dual component signal system in the embryo sac. The analysis of the promoter activity of all 7 histidine kinase genes involved in the double component signal transduction of the 7 histidine kinase genes in the embryo sac development showed that the expression signals were detected in the embryo sac, including AHK1, AHK3, AHK4, AHK5, CKI1 and ETRl in the embryo sac, and the expression of CKI1 was relatively high. The observation of AHK4 three mutant showed that AHK2, AHK3 and AHK4 had functional redundancy in regulating the development of the functional megaspore. (2) the ckil-9/+ mutant of Arabidopsis showed the abortive phenotype of the female gametophyte. The observation of the ovule DIC showed that the two polar nuclei in the mutant embryo sac often did not merge normally, and were accompanied by three anti degradation degradation. The cell nuclei migrated from the end of the embryo sac to the end of the pearl hole. The single 1narker and double marker expressed by the female gametophyte cells were constructed, and the marker was transferred into the ckil-9, / + mutant by the Arabidopsis thaliana transformation method. The results showed that the fate of the antipodal and Central cells in the mutant embryo sac was completely lost, and three antipodal cells were lost. And two non fused polar nuclei show the fate of the egg cells or the cells of the helper cells. Location analysis of the CKI1 protein in the development of the embryo sac indicates that CKIl begins to show the polar location of the junction end from the FG4 period of the embryo sac development, which may be realized through the regulation mechanism after the gene transcription. Using the ES1 promoter. The CKIl gene was overexpressed in the embryo sac, and the results showed that the ectopic expression of CKI1 in the egg and the helper cells could transform the fate of the egg and the helper cells into the cell fate of the central cell. Based on the above results, two models of the fate of the female gametocyte cells were proposed. (3) the promoter activity of IHPI ~ AHP5 in the mature embryo sac. The results showed that the AHP1 gene was expressed in the central and helper cells, and the AHP3 gene was expressed specifically in the central cells. The AHP2 and AHP5 genes were expressed in the central, oocyte, helper and antipodal cells, and the AHP4 gene was not expressed in the embryo sac. Then, the multiple AHP mutants were constructed and the seed development and embryo of the multiple mutants were developed. The results showed that the ahp2-2 ahp3 ahp5-2/+ three mutant showed a similar phenotype to ckil-9/+, and AHP2, AHP3 and AHP5 co regulated the development of the female gametophyte in the lower reaches of the CKIl gene. (4) the comparison of the amino acid sequence of the plant HK family protein found that CKI1 existed in the HK domain and the HATPase domain respectively. The base acid sequence is a specific motif of SHD and GLGLG, which can be used as an important basis for determining CKI1. The relationship between the domain of CKI1 and the function of CKI1 gene is studied by the replacement of CKI1 domain by overlap extension PCR technology. The results of the localization of white subcells show that the N- terminal region of CKI1 determines the localization of CKI1 protein on the endoplasmic reticulum. (5) the study of different lengths of CKI1 promoter indicates that the core promoter element of CKIl is located in the first intron. Using yeast single heterozygosity to screen and identify the upstream transcriptional regulators of the CKI1 and the final screening. 7 transcriptional factor genes expressed in central cells were selected. (6) 13 IPTs and 12 CKXs genes were identified in Chinese cabbage genome, and their genomic information and protein characteristics were analyzed. Multiple sequence alignment, conservative domain and phylogenetic analysis showed that both IPTs and CKXs can be divided into I, II, and III. A comparative analysis of IPTs and CKXs in cabbage, Arabidopsis and three cruciferous plants of Arabidopsis thaliana from Chinese cabbage, Arabidopsis, and Arabidopsis thaliana was made by comparison and analysis of genomic level. It was found that the gene existed in fragment repetition and series duplication, and the evolution patterns of the genes were analyzed by calculating the Ks and Ka values of the direct homologous and accessory homologous genes. The results showed that the Ks value of the homologous genes in Chinese cabbage was obviously smaller than that of Chinese cabbage, Arabidopsis and Arabidopsis thaliana in Arabidopsis thaliana. At the same time, Chinese cabbage was separated from Arabidopsis in about 26 to 33MYA. In addition, some organ specific tables were screened by qRT-PCR technology to analyze the pattern of IPTs and CKXs in different organs of Chinese cabbage. The genes of IPTs and CKXs were studied under drought stress and salt stress, and the response patterns of external cytokinin and abscisic acid were studied. Some candidate genes involved in the abiotic stress and hormone regulation network were obtained. (7) a total of 85 TCS gene members were identified by identification of two component signal components in Chinese cabbage. 20 HK genes, 8 HPs and 57 RRs were included, and their genomic information and protein characteristics were analyzed. Multiple sequence alignment, conservative domain and phylogenetic analysis showed that HKs could be divided into the cytokinin subfamily, the AHKl subfamily, the AHK5 subfamily, the CKI1 subfamily, the ethylene receptor subfamily and the photosensitive subfamily.RRs. It is divided into four categories: Type-A RR, Type-B RR, Type-C RR and pseudo-RR. We analyzed and compared the repeat and tandem repeats of TCS gene in Chinese cabbage and Arabidopsis, and analyzed the evolution patterns of the gene by calculating the Ks and Ka values of the direct homologous and accessory homologous genes. The expression patterns in the same organs were analyzed, and some organ specific genes were screened. In addition, the response characteristics of Chinese cabbage TCSs to drought stress and salt stress, as well as the response characteristics of external cytokinin, auxin and abscisic acid were also studied. (8) 281 AP2/ERF supergene families were identified in the cabbage genome. The analysis shows that the conserved domain and the evolutionary tree are analyzed. The results show that the AP2/ERF superfamily can be divided into AP2 family, RAV family and ERF family, and the ERF family can be further divided into IREB subfamily and ERF subfamily, including 13 groups I to ERF, and 21 CRFs in group VI and VI-L. It can be divided into 5 branches of I to 5, encoding three types of protein, A, B and C. Using qRT-PCR technology to study the expression patterns of CRFs in different organs. The results show that CRFs is expressed in different organs, but there are certain differences in the expression of different parts. Through the study of CRFs response to drought stress and salt stress As well as the response patterns of external cytokinin and abscisic acid, a number of candidate genes involved in the network of abiotic stress and hormone regulation are screened. The results can enable us to understand the development of the female gametophyte in plants from a new perspective, and to fully understand the molecular mechanism of sexual reproduction of the economic crops, and improve the molecular mechanism of the sexual reproduction of the economic crops. The research foundation of reproductive biology of Brassica vegetable crops provides a new theoretical basis and technical support for effectively regulating crop fertility and achieving high quality, high yield and high efficiency reproduction.
【學(xué)位授予單位】：浙江大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：Q944.4;Q943.2
，

本文編號：1995473