本文選題：擬南芥 + 減數(shù)分裂�。� 參考：《中國農(nóng)業(yè)大學(xué)》2017年博士論文

【摘要】：在顯花植物(phanerogams)的有性生殖過程中,雌蕊胚珠和雄蕊花藥中的性母細(xì)胞(2n)經(jīng)減數(shù)分裂分別形成大孢子(n)和小孢子(n)后,進(jìn)一步發(fā)育分別形成雌配子體(胚囊)和雄配子體(花粉)。減數(shù)分裂是雌、雄配子體形成的關(guān)鍵步驟,包括DNA復(fù)制、姐妹染色單體黏連、同源染色體配對、聯(lián)會、重組和分離等一系列重要的生物學(xué)事件,任何環(huán)節(jié)出現(xiàn)異常,都會影響雌雄配子體的形成,導(dǎo)致植物不育。其中,同源染色體重組是遺傳信息交換的重要方式,而DNA雙鏈斷裂(DNA double-strand break,DSB)的形成是同源重組起始過程,目前已發(fā)現(xiàn)幾個蛋白參與調(diào)控?cái)M南芥DSB的形成,其中AtSPO11-1和AtSP011-2是SPO11的同源蛋白;AtPRD1是與小鼠中MEI1同源但功能未知的蛋白;AtPRD2是小鼠和酵母中Mei4的同源蛋白;AtPRD3和AtDFO是植物所特有但功能未知的DSB形成蛋白。雖然已發(fā)現(xiàn)這幾個蛋白參與DSB形成過程,但對DSB形成過程的遺傳調(diào)控機(jī)制還缺乏深入了解。因此,研究植物雌、雄配子體的形成以及同源染色體重組的分子遺傳機(jī)制不僅可以了解植物生殖發(fā)育的分子遺傳機(jī)制,也可為農(nóng)作物遺傳育種提供遺傳學(xué)方面的理論參考。本研究分離鑒定到了影響擬南芥雌雄配子體發(fā)育的突變體mt187,該突變體是一個從基因陷阱(gene-trap)和增強(qiáng)子陷阱(enhancer-trap)Ds插入突變體庫中篩選到的。與野生型相比,它的角果短小,育性降低。遺傳分析顯示,mt187突變體中的Ds插入位點(diǎn)與表型不連鎖。圖位克隆技術(shù)分析顯示,突變位點(diǎn)位于At1g60460基因的第八個外顯子的最后一個堿基(G1890),該位點(diǎn)G1890A突變造成相關(guān)內(nèi)含子的剪切異常,從而影響該基因的正常表達(dá),并嚴(yán)重影響雌雄配子體的形成,導(dǎo)致植物的育性顯著降低。另外,從擬南芥生物資源中心(ABRC)獲得的T-DNA插入等位突變體也具有相似的表型。這些研究結(jié)果表明,At1g60460基因在雌雄配子體的形成中起重要作用。At1g60460基因編碼一個最近被命名為MTOPVIB的蛋白。鑒于已有兩個相關(guān)的等位突變體,因此把mt187改名為mtopVIB-3。上述T-DNA插入等位突變體與發(fā)表在Science中的mtopVIB-2是同一個突變體。相對于mtopVIB-3,T-DNA插入突變體mtopVIB-2具有更嚴(yán)重的表型,其結(jié)實(shí)率僅有2%。初步觀察發(fā)現(xiàn),不僅mtopVIB突變體花藥中的四分體發(fā)育異常、花粉敗育,而且它的胚珠中的雌配子體發(fā)育也異常,停滯在FG1期大孢子母細(xì)胞時期,暗示可能是雌雄配子體發(fā)育過程中的減數(shù)分裂過程出現(xiàn)異常。進(jìn)一步利用染色體展片方法觀察發(fā)現(xiàn),與野生型的雄和雌性母細(xì)胞減數(shù)分裂相比,在粗線期,mtopVIB突變體的染色體沒有呈現(xiàn)正常的粗絲狀;在終變期,突變體出現(xiàn)單價染色體,而不是正常的五對二價體。同時通過FISH實(shí)驗(yàn)和免疫熒光實(shí)驗(yàn)證明,突變體的染色體配對、聯(lián)會出現(xiàn)異常。通過計(jì)算突變體的重組率,發(fā)現(xiàn)突變體的重組率較野生型顯著地下降,表明mtopVIB突變體的同源重組也異常。將表型嚴(yán)重的mtopVIB-2突變體與DSB修復(fù)缺陷突變體Atcom1-1、Atrad50-1、Atrad51和Atmre11-1構(gòu)建相應(yīng)的雙突變體,這些雙突變體的表型與mtopVIB-2突變體的表型相似,均在減數(shù)分裂過程中出現(xiàn)單價體,表明mtopVIB-2確實(shí)影響DSB的形成,說明MTOPVIB參與減數(shù)分裂DSB的形成過程。Real-time PCR分析表明,MTOPVIB呈組成型表達(dá),在花序中表達(dá)量較高。MTOPVIB蛋白屬于一類保守性較高的蛋白,其結(jié)構(gòu)與Topo VI型復(fù)合體的B亞基相似,其N端含有一個可以結(jié)合并水解ATP的Bergerat結(jié)構(gòu)域,C端含有一個可以誘導(dǎo)構(gòu)象變化的Transducer結(jié)構(gòu)域。Y2H和BiFC實(shí)驗(yàn)結(jié)果表明,MTOPVIB蛋白可以分別與AtSPO11-1和AtSPO11-2蛋白互作,而且Y3H和BiFC實(shí)驗(yàn)顯示,MTOPVIB蛋白可以同時與它們互作,暗示三者間可能形成一個Topo VI類似復(fù)合體。Y2H和BiFC實(shí)驗(yàn)還表明,AtPRD1可以分別與MTOPVIB和AtSP011-2互作,說明AtPRD1可以與Topo VI類似復(fù)合體的各成員互作。利用同樣的方法還發(fā)現(xiàn)AtPRD1蛋白還可以分別與AtPRD3和AtDFO蛋白互作。此外,Y3H和BiFC實(shí)驗(yàn)顯示AtPRD1蛋白可以介導(dǎo)與AtPRD3和AtDFO蛋白的互作。這表明AtPRD1很可能是DSB形成過程中的一個重要蛋白。綜上所述,在擬南芥中,MTOPVIB蛋白與AtPRD1蛋白互作,并與AtSPO11-1、AtSP011-2、AtPRD3和AtDFO等蛋白共同參與擬南芥雌雄配子體減數(shù)分裂過程的DSB形成過程。這些結(jié)果為了解擬南芥減數(shù)分裂DSB形成的機(jī)制提供新的實(shí)驗(yàn)證據(jù)。
[Abstract]:In the sexual reproduction of phanerogams, the female gametophyte (n) and microspore (n) in the anther of pistil ovule and stamen anther are formed to form a female gametophyte (embryo sac) and male gametophyte (pollen) respectively. Meiosis is a key step in the formation of female and male gametophytes, including DNA replication. A series of important biological events, such as sister chromatid adhesion, homologous chromosome pairing, association, recombination and separation, can affect the formation of male and male gametophytes and cause plant infertility. Among them, homologous chromosome recombination is an important way of genetic information exchange, and DNA double strand breaks (DNA double-strand break, DSB) ) formation is the initiation process of homologous recombination. Several proteins have been found to regulate the formation of Arabidopsis DSB, in which AtSPO11-1 and AtSP011-2 are SPO11 homologous proteins; AtPRD1 is the homologous protein of MEI1 in mice but unknown in function; AtPRD2 is the same source protein of Mei4 in mice and yeast; AtPRD3 and AtDFO are endemic but not functional of plants. The DSB formation protein is known. Although these proteins have been found to be involved in the formation of DSB, the genetic regulation mechanism for the formation of DSB is still lack of understanding. Therefore, the molecular genetic mechanism of the study on the formation of female and male gametophytes and the recombination of homologous chromosomes can not only be used to understand the molecular genetic mechanism of plant reproductive development, but also can be used for the study of the molecular genetic mechanism of plant reproductive development. Genetic breeding for crop genetics is a theoretical reference. This study identified a mutant mt187 that affects the development of male and female gametophyte development in Arabidopsis. This mutant is screened from the gene trap (gene-trap) and enhancer trap (enhancer-trap) Ds insertion mutant library. Compared with the wild type, the mutant is short and fertile. The genetic analysis showed that the Ds insertion site in the mt187 mutant was not linked to the phenotype. The genetic analysis showed that the mutation site was located at the last base of the eighth exons of the At1g60460 gene (G1890), and the G1890A mutation caused the shear anomaly of the related introns, which affected the normal expression of the gene and was serious. In addition, the T-DNA insertion alleles obtained from the Arabidopsis Biological Resource Center (ABRC) also have similar phenotypes. These results suggest that the At1g60460 gene plays a role in the formation of the female and male gametophyte to act as the.At1g60460 gene encoding one recently named as MTOPVIB protein, in view of two related alleles, mt187 was renamed mtopVIB-3. as the same T-DNA insertion mutant and mtopVIB-2 published in Science. Relative to mtopVIB-3, T-DNA insertion mutant mtopVIB-2 has a more serious form, and its seed setting rate is only observed by 2%.. Only four of the mtopVIB mutant anthers have abnormal development, pollen abortion, and the development of the female gametophyte in the ovule is also abnormal, stagnating in the period of the FG1 stage of the megaspore mother cell, suggesting that the meiotic division during the development of the male and the male gametophyte may be abnormal. Compared with the male and female meiosis meiosis, the chromosomes of the mtopVIB mutant did not present normal coarse filamentous chromosomes at the roughing stage. At the final stage, the mutants appeared monovalent chromosomes, but not the normal five to two valence bodies. At the same time, the chromosomal pairing of the mutants appeared to be abnormal through the FISH experiment and immunofluorescence experiments. The recombination rate of the mutant showed that the recombinant rate of the mutant was significantly lower than that of the wild type, indicating that the homologous recombination of the mtopVIB mutant was also abnormal. The phenotype of the mutant mtopVIB-2 and the DSB repair defect mutants Atcom1-1, Atrad50-1, Atrad51 and Atmre11-1 were constructed, and the phenotype of these double mutants and the mtopVIB-2 process were the same. The phenotypic similarity of the variant is similar to that of the monovalent in the meiosis process, indicating that mtopVIB-2 does affect the formation of DSB, indicating that MTOPVIB participates in the formation of meiotic DSB,.Real-time PCR analysis indicates that MTOPVIB is formed in group form, and that the higher expression of.MTOPVIB in the inflorescence belongs to a class of conserved protein, and its structure and To The B subunit of the Po VI complex is similar, and its N terminal contains a Bergerat domain that can bind and hydrolyze ATP. The C end contains a Transducer domain that can induce conformation changes,.Y2H and BiFC experimental results show that MTOPVIB protein can be interacted with AtSPO11-1 and protein respectively. At the same time interacting with them, it is suggested that a Topo VI similar complex.Y2H and BiFC experiment may be formed among the three, and that AtPRD1 can interact with MTOPVIB and AtSP011-2 respectively, indicating that AtPRD1 can interact with the members of the Topo VI similar complex. In addition, Y3H and BiFC experiments show that AtPRD1 protein can mediate interaction with AtPRD3 and AtDFO proteins. This indicates that AtPRD1 is likely to be an important protein in the formation of DSB. To sum up, in Arabidopsis, MTOPVIB protein is interacted with AtPRD1 protein and participates in Arabidopsis together with AtSPO11-1, AtSP011-2, AtPRD3, and such proteins. DSB results in the process of gametophyte meiosis. These results provide new experimental evidence for understanding the mechanism of meiosis DSB formation in Arabidopsis thaliana.

【學(xué)位授予單位】：中國農(nóng)業(yè)大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2017
【分類號】：Q943.2

【參考文獻(xiàn)】

相關(guān)期刊論文 前2條

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2 楊克珍;葉德;;植物雄配子體發(fā)生和發(fā)育的遺傳調(diào)控[J];植物學(xué)通報(bào);2007年03期

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本文編號：1848728