本文選題：水稻 + 減數(shù)分裂��； 參考：《南京農(nóng)業(yè)大學(xué)》2016年博士論文

【摘要】：水稻是全球最主要的糧食作物之一,世界上一半以上的人口以稻米為主食,在我國比例更是高達(dá)60%以上·目前,在我國人口數(shù)量不斷增加,而耕地面積不斷減少的形勢(shì)下,通過大幅度提高單位土地面積的糧食產(chǎn)量是保障我國糧食安全的唯一途徑。二十世紀(jì)七十年代中期雜交水稻的成功培育,是世界農(nóng)業(yè)發(fā)展史上的一次重大突破,被譽(yù)為繼水稻矮化育種之后的“第二次綠色革命”,創(chuàng)造了巨大的經(jīng)濟(jì)效益和社會(huì)效益。然而,統(tǒng)計(jì)數(shù)據(jù)表明近些年水稻單產(chǎn)的提高潛力已經(jīng)十分有限。其主要原因一方面是配制雜交組合的親本遺傳資源缺乏,親本間遺傳差異小,導(dǎo)致雜種優(yōu)勢(shì)不強(qiáng);另一方面是種間、亞種間雜種優(yōu)勢(shì)強(qiáng)大,但普遍存在生殖隔離等問題,嚴(yán)重限制了雜種優(yōu)勢(shì)的直接利用。因此,探索雄性不育,種間生殖隔離機(jī)理對(duì)進(jìn)一步利用雜種優(yōu)勢(shì)具有重要的理論價(jià)值和實(shí)踐意義。水稻雜種優(yōu)勢(shì)的利用最早是從發(fā)現(xiàn)一株典型花粉敗育野生稻獲得突破的。典敗型花粉的產(chǎn)生是由于花粉不能正常進(jìn)行減數(shù)分裂導(dǎo)致的,因此,了解水稻減數(shù)分裂分子機(jī)理能更好的為雜種優(yōu)勢(shì)利用提供理論指導(dǎo)。研究表明,非洲栽培稻經(jīng)過長時(shí)間的人為選擇進(jìn)化,聚集了很多亞洲栽培稻缺乏的優(yōu)良特性,如抗蟲、抗病、抗逆以及旺盛的生長優(yōu)勢(shì)。因此,為了豐富水稻的遺傳資源,提高水稻產(chǎn)量,打破育種瓶頸,將非洲栽培稻優(yōu)異基因?qū)氲絹喼拊耘嗟臼沁h(yuǎn)緣雜種優(yōu)勢(shì)利用的首選。然而,亞洲栽培稻和非洲栽培稻的雜交F1存在嚴(yán)重的生殖隔離,如何深入了解并克服雜種不育是有效利用栽培稻種間雜種優(yōu)勢(shì)的前提。本論文分兩個(gè)方面對(duì)水稻雄性不育進(jìn)行了研究。首先,通過一個(gè)水稻雄性不育突變體克隆了水稻OsMSH4基因,對(duì)其功能進(jìn)行了研究;其次,精細(xì)定位了種間雜種花粉不育基因S39并對(duì)其花粉敗育過程進(jìn)行了詳細(xì)觀察。本研究的主要內(nèi)容如下:1.從水稻四倍體花藥組織培養(yǎng)后代中,我們發(fā)現(xiàn)一株三體植株6537。該植株與正常植株相比葉色更深,葉片變窄且伴有卷曲,染色體壓片觀察發(fā)現(xiàn)其多了一條染色體,證明6537是典型的三體植株。從6537的自交后代中,分離出大約有3/4的完全不育株,暫命名為Osmsh4突變體。用I2-KI溶液對(duì)突變體的花粉進(jìn)行染色,觀察顯示其不能被染色。掃描電鏡觀察顯示突變體的花粉粒大小不均一,且形狀不規(guī)則;透射電鏡觀察發(fā)現(xiàn)其內(nèi)部沒有淀粉粒的積累,花粉粒壁的發(fā)育也不完全,從而導(dǎo)致Osmsh4突變體的花粉是完全敗育的。利用激光共聚焦掃描電鏡對(duì)Osmsh4突變體的胚囊發(fā)育進(jìn)行觀察發(fā)現(xiàn)其不能進(jìn)行正常的減數(shù)分裂形成四分體,以致喪失功能大孢子的分化,沒有七細(xì)胞八核正常胚囊形成,最終導(dǎo)致Osmsh4突變體的胚囊也敗育。2.花粉母細(xì)胞染色體減數(shù)分裂觀察發(fā)現(xiàn)在粗線期及其之前,Osmsh4突變體和野生型在染色體行為上沒有明顯的差別,然而在雙線期,Osmsh4突變體花粉母細(xì)胞中開始有單價(jià)體的出現(xiàn),到了終變期這種單價(jià)體數(shù)目逐漸增多,且尤為明顯。從后期Ⅰ到末期Ⅱ,由于單價(jià)體的隨機(jī)分離,導(dǎo)致移動(dòng)到兩極的染色體數(shù)不均等。這種不均等勢(shì)必會(huì)導(dǎo)致隨后形成的小孢子體內(nèi)染色體組的紊亂,產(chǎn)生三分體或者微核。與野生型12條二價(jià)體相比,Osmsh4突變體平均只有3.95條。由于交叉結(jié)是穩(wěn)定二價(jià)體的保障,因此突變體中二價(jià)體數(shù)目的減少很可能是交叉結(jié)數(shù)目的減少引起的。統(tǒng)計(jì)發(fā)現(xiàn)野生型平均有20.58個(gè)交叉結(jié)(n = 81),而Osmsh4突變體只有4.51個(gè)(n= 152)。而且突變體中剩余交叉的分布是隨機(jī)的,與泊松分布相吻合;而野生型中的交叉結(jié)分布嚴(yán)重偏離泊松分布,說明野生型中的交叉結(jié)之間有干涉的存在,而Osmsh4突變體中剩余的交叉結(jié)之間則沒有干涉存在。3.通過圖位克隆我們克隆了 OsMSH4基因。序列比對(duì)發(fā)現(xiàn)OsMSH4蛋白在其它物種中都是保守的,蛋白分析顯示其有一個(gè)MUTSd和MUTSac結(jié)構(gòu)域,而Osmsh4突變體的單氨基酸替換正好發(fā)生在保守的MUTSd結(jié)構(gòu)域上。我們通過轉(zhuǎn)基因互補(bǔ)實(shí)驗(yàn)以及對(duì)OsMSH4基因TOS17突變體的調(diào)查證實(shí)OsMSH4就是目的基因。熒光定量、GUS染色和組織原位雜交分析表明,OsMSH4主要在生殖器官中表達(dá),而且呈現(xiàn)一種動(dòng)態(tài)變化,在減數(shù)分裂時(shí)期花藥的性母細(xì)胞中表達(dá)最高,隨后表達(dá)水平逐漸降低直至消失。亞細(xì)胞定位顯示OsMSH4蛋白主要定位在細(xì)胞核中,與其功能相一致。4.前人研究表明MSH4蛋白能和MSH5蛋白一起互作形成異源二聚體結(jié)合到Holliday連接體上來穩(wěn)定單鏈入侵,通過酵母雙雜交和體外pull-down實(shí)驗(yàn)我們證明OsMSH4也能和OsMSH5互作,而且突變后的Osmsh4就喪失了與OsMSH5的互作能力;此外,我們還發(fā)現(xiàn)OsMSH5能夠與OsRPA蛋白復(fù)合體中的OsRPA1a, OsRPA2b,OsRPA1c和OsRPA2c四個(gè)亞基互作。因?yàn)镺sRPA復(fù)合體是結(jié)合到D環(huán)和單鏈DNA上的,說明在減數(shù)分裂過程中,OsMSH4/OsMSH5異源二聚體與OsRPA復(fù)合體互作來共同調(diào)控 Holliday 交叉(Holliday junction)第二鏈的捕獲(second-end capture, SEC )。5.利用以亞洲栽培稻滇粳優(yōu)1號(hào)(DJY)為受體,非洲栽培稻IRGC102295為供體構(gòu)建的近等基因系(near-isogenic lines, NIL),我們鑒定了種間雜種花粉不育基因S39。細(xì)胞學(xué)觀察發(fā)現(xiàn)雜種F1的花粉表現(xiàn)為半不育,屬于染敗,敗育的花粉粒比正常的偏小且形狀不規(guī)則,淀粉積累少,花粉粒壁發(fā)育不完全。進(jìn)一步的研究發(fā)現(xiàn)F1中敗育的花粉粒主要為DJY類型,敗育時(shí)期發(fā)生在小孢子中期向晚期轉(zhuǎn)變的過程,由于此過程的滯后導(dǎo)致DJY型的配子不能形成正常的三核花粉,淀粉積累也不完全。6.利用次級(jí)分離群體我們將S39定位在第12號(hào)染色體79-kb的區(qū)間,該區(qū)域一共預(yù)測(cè)了 13個(gè)候選基因。對(duì)該區(qū)間進(jìn)行測(cè)序發(fā)現(xiàn)兩親本序列存在較大的差異,其中NIL比親本DJY多了約82-kb的序列。進(jìn)一步分析發(fā)現(xiàn)S39位于一個(gè)400-kb左右的品種間相互倒位區(qū)間。熒光定量結(jié)果顯示定位區(qū)間幾個(gè)基因在花藥中高表達(dá),然而單個(gè)基因的轉(zhuǎn)基因?qū)嶒?yàn)卻沒有得到互補(bǔ)表型。遺傳分析發(fā)現(xiàn)S39位點(diǎn)是一個(gè)普遍存在于種間和亞種間的雜種不育位點(diǎn),而廣親和品種Dular,Kasalath在此位點(diǎn)攜帶有親和基因。
[Abstract]:Rice is one of the most important food crops in the world. More than half of the population in the world takes rice as the staple food. The proportion of rice is more than 60% in our country. At present, in the situation of increasing population in China and the continuous reduction of the cultivated land area, the grain yield of the unit soil area is guaranteed by a large margin, which guarantees the food security of our country. The only way. The successful cultivation of hybrid rice in the middle of the 1970s is a major breakthrough in the history of world agricultural development. It is known as the "second Green Revolution" after rice dwarfing breeding, which has created enormous economic and social benefits. However, the statistical data show that the potential for increasing rice yield in recent years has already been improved. The main reasons are the lack of parental genetic resources for the preparation of hybrid combinations, small genetic differences between parents and poor heterosis; on the other hand, the heterosis is strong between species and subspecies, but the common existence of reproductive isolation is common, which severely restricts the direct use of heterosis. Therefore, male infertility is explored. The mechanism of inter reproductive isolation is of great theoretical and practical significance for further utilization of heterosis. The earliest use of Heterosis in rice is a breakthrough in the discovery of a typical pollen abortion wild rice. The production of canonical pollen is caused by the failure of the pollen to carry out the normal meiosis. Therefore, the meiosis of rice is understood. The molecular mechanism can better provide theoretical guidance for the use of heterosis. Studies have shown that a long period of artificial selection of cultivated rice in Africa has gathered a lot of excellent characteristics, such as insect resistance, disease resistance, resistance, and strong growth advantages. Therefore, it has enriched rice genetic resources and improved rice yield. Breeding bottlenecks, introducing African cultivated rice genes into Asian cultivated rice is the first choice for heterosis. However, the hybrid F1 between cultivated and African cultivated rice has serious reproductive isolation. How to understand and overcome hybrids is a prerequisite for the effective utilization of Hybrid Heterosis in cultivated rice. This paper is divided into two aspects. The male sterility of rice was studied. First, the rice OsMSH4 gene was cloned through a rice male sterile mutant, and its function was studied. Secondly, the interspecific hybrid pollen sterile gene S39 was carefully located and its pollen abortion process was observed in detail. The main contents of this study are as follows: 1. from the tetraploid rice flower of rice In the progeny of drug tissue culture, we found that a trisomy plant 6537. had a deeper leaf color, a narrower leaf and a curly leaf compared with the normal plant. The chromosome compression observation found that the plant was more than one chromosome, proving that 6537 was a typical trisomy plant. From 6537 of the self bred progeny, a complete sterile plant of about 3/4 was separated, temporarily named Osm SH4 mutant. The pollen of the mutant was stained with I2-KI solution, and the observation showed that it could not be dyed. The scanning electron microscope showed that the size of the pollen grains was not uniform and the shape was irregular. The transmission electron microscope observed that there was no accumulation of starch grains in the mutant and the development of the pollen grain wall was incomplete, thus leading to the flower of the Osmsh4 mutant. The powder was completely aborted. Using laser confocal scanning electron microscopy to observe the development of the embryo sac of the Osmsh4 mutant, it was found that it could not carry out the normal meiosis to form four division, so that the differentiation of the functional megaspore, no seven cells and eight nucleus normal embryo sac were formed, and the embryo sac of the Osmsh4 mutant was eventually aborted by the.2. pollen mother cell. Chromosome meiosis observation showed that there was no obvious difference in chromosome behavior between the Osmsh4 mutants and the wild type before the rough line period and the wild type. However, in the biphase, the Osmsh4 mutant of the pollen mother cell began to appear in the monovalent body, and the number of the monovalent in the final stage was gradually increasing and particularly obvious. As a result of the random separation of the monovalent, the number of chromosomes moving to the poles is uneven. This inequality is bound to lead to the disorder of the chromosomes in the microspores that are subsequently formed, producing three bodies or micronucleus. Compared with the wild type 12 two valence bodies, the average of the Osmsh4 mutants is only 3.95. Because the intersection is the guarantee of the stable two valence body, Therefore, the reduction of the number of two valence bodies in the mutant was likely to be caused by the reduction of the number of cross knot numbers. The statistics found that there were 20.58 cross junctions (n = 81) on the average of the wild type, but only 4.51 (n= 152) of the Osmsh4 mutant, and the distribution of the remaining crosses in the mutant was random and consistent with the distribution of Poisson; and the cross knot in the wild type was strictly distributed. A heavy deviation from the Poisson distribution shows that there is interference between the intersections of the wild type, while the remaining junctions in the Osmsh4 mutants have no interference in the presence of.3. and we cloned the OsMSH4 gene through the map. The sequence alignment found that the OsMSH4 protein is kept in other species, and the protein analysis shows that it has a MUTSd and MUTS. The AC domain, while the single amino acid substitution of the Osmsh4 mutant occurs just in the conservative MUTSd domain. We confirm that OsMSH4 is the target gene through the transgene complementarity experiment and the investigation of the OsMSH4 gene TOS17 mutants. Fluorescence quantitative, GUS staining and tissue in situ hybridization analysis show that OsMSH4 is mainly expressed in the reproductive organs. The expression of the anther in the anther is the highest in the meiotic stage of the meiosis, and then the expression level gradually decreases to the disappearance. The subcellular localization shows that the OsMSH4 protein is mainly located in the nucleus, and its function is consistent with the function of.4.. The previous study indicates that the MSH4 protein can interact with the MSH5 protein to form the allogeneic two polymer. In the Holliday connection to stabilize the single strand invasion, by yeast two hybrid and in vitro pull-down experiments, we have shown that OsMSH4 can also interact with OsMSH5, and the mutant Osmsh4 loses its ability to interact with OsMSH5; furthermore, we also found that OsMSH5 can be associated with the four subtypes of OsRPA1a, OsRPA2b, OsRPA1c, and OsRPA2c in the OsRPA protein complex. Because the OsRPA complex is combined with the D ring and the single strand DNA, it is indicated that during the meiosis, the OsMSH4/OsMSH5 heterogenous two polymer and the OsRPA complex interacted together to regulate the capture of the Holliday Cross (Holliday junction) second chain (second-end capture, SEC).5., using the Asian cultivated rice Dian Jing you 1 as a receptor, Chau cultivated rice IRGC102295 was a near isogenic line (near-isogenic lines, NIL) constructed from the donor. We identified the S39. cytology of the interspecific hybrid pollen sterile gene by S39. cytology. It was found that the pollen of hybrid F1 was semi sterile, which belonged to dyed and aborted, and the aborted pollen grains were smaller and irregular than normal, and the accumulation of starch was less, and the pollen grain wall development was not complete. Further studies have found that the pollen grains aborted in F1 are mainly DJY type, and the period of abortion at the middle of the microspore to the late stage of microspore transformation, because the lag of this process causes the DJY type gamete to not form normal three nuclear pollen, and the accumulation of starch is not completely.6. using the secondary segregating group, and we locate S39 on chromosome twelfth 79-. A total of 13 candidate genes were predicted in the region of the KB. The sequence of the two parent sequences was found to be different, in which the sequence of NIL was more than 82-kb in the parent DJY. Further analysis found that S39 was located in the reciprocal interval of a 400-kb variety. The fluorescence quantitative results showed that several genes were in the location interval. The anthers are highly expressed, however, the single gene has not been complemented by the transgenic experiment. The genetic analysis found that the S39 site is a hybrid sterile site commonly found in interspecific and subspecific, while the wide compatible Dular, Kasalath at this site carries an affinity gene.

【學(xué)位授予單位】：南京農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：S511

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