水稻减数分裂基因OsMSH4的功能研究和种间杂种不育基因S39的精细定位

发布时间：2018-05-13 04:11

本文选题：水稻 + 减数分裂　；参考：《南京农业大学》2016年博士论文

【摘要】：水稻是全球最主要的粮食作物之一,世界上一半以上的人口以稻米为主食,在我国比例更是高达60%以上·目前,在我国人口数量不断增加,而耕地面积不断减少的形势下,通过大幅度提高单位土地面积的粮食产量是保障我国粮食安全的唯一途径。二十世纪七十年代中期杂交水稻的成功培育,是世界农业发展史上的一次重大突破,被誉为继水稻矮化育种之后的“第二次绿色革命”,创造了巨大的经济效益和社会效益。然而,统计数据表明近些年水稻单产的提高潜力已经十分有限。其主要原因一方面是配制杂交组合的亲本遗传资源缺乏,亲本间遗传差异小,导致杂种优势不强;另一方面是种间、亚种间杂种优势强大,但普遍存在生殖隔离等问题,严重限制了杂种优势的直接利用。因此,探索雄性不育,种间生殖隔离机理对进一步利用杂种优势具有重要的理论价值和实践意义。水稻杂种优势的利用最早是从发现一株典型花粉败育野生稻获得突破的。典败型花粉的产生是由于花粉不能正常进行减数分裂导致的,因此,了解水稻减数分裂分子机理能更好的为杂种优势利用提供理论指导。研究表明,非洲栽培稻经过长时间的人为选择进化,聚集了很多亚洲栽培稻缺乏的优良特性,如抗虫、抗病、抗逆以及旺盛的生长优势。因此,为了丰富水稻的遗传资源,提高水稻产量,打破育种瓶颈,将非洲栽培稻优异基因导入到亚洲栽培稻是远缘杂种优势利用的首选。然而,亚洲栽培稻和非洲栽培稻的杂交F1存在严重的生殖隔离,如何深入了解并克服杂种不育是有效利用栽培稻种间杂种优势的前提。本论文分两个方面对水稻雄性不育进行了研究。首先,通过一个水稻雄性不育突变体克隆了水稻OsMSH4基因,对其功能进行了研究;其次,精细定位了种间杂种花粉不育基因S39并对其花粉败育过程进行了详细观察。本研究的主要内容如下:1.从水稻四倍体花药组织培养后代中,我们发现一株三体植株6537。该植株与正常植株相比叶色更深,叶片变窄且伴有卷曲,染色体压片观察发现其多了一条染色体,证明6537是典型的三体植株。从6537的自交后代中,分离出大约有3/4的完全不育株,暂命名为Osmsh4突变体。用I2-KI溶液对突变体的花粉进行染色,观察显示其不能被染色。扫描电镜观察显示突变体的花粉粒大小不均一,且形状不规则;透射电镜观察发现其内部没有淀粉粒的积累,花粉粒壁的发育也不完全,从而导致Osmsh4突变体的花粉是完全败育的。利用激光共聚焦扫描电镜对Osmsh4突变体的胚囊发育进行观察发现其不能进行正常的减数分裂形成四分体,以致丧失功能大孢子的分化,没有七细胞八核正常胚囊形成,最终导致Osmsh4突变体的胚囊也败育。2.花粉母细胞染色体减数分裂观察发现在粗线期及其之前,Osmsh4突变体和野生型在染色体行为上没有明显的差别,然而在双线期,Osmsh4突变体花粉母细胞中开始有单价体的出现,到了终变期这种单价体数目逐渐增多,且尤为明显。从后期Ⅰ到末期Ⅱ,由于单价体的随机分离,导致移动到两极的染色体数不均等。这种不均等势必会导致随后形成的小孢子体内染色体组的紊乱,产生三分体或者微核。与野生型12条二价体相比,Osmsh4突变体平均只有3.95条。由于交叉结是稳定二价体的保障,因此突变体中二价体数目的减少很可能是交叉结数目的减少引起的。统计发现野生型平均有20.58个交叉结(n = 81),而Osmsh4突变体只有4.51个(n= 152)。而且突变体中剩余交叉的分布是随机的,与泊松分布相吻合;而野生型中的交叉结分布严重偏离泊松分布,说明野生型中的交叉结之间有干涉的存在,而Osmsh4突变体中剩余的交叉结之间则没有干涉存在。3.通过图位克隆我们克隆了 OsMSH4基因。序列比对发现OsMSH4蛋白在其它物种中都是保守的,蛋白分析显示其有一个MUTSd和MUTSac结构域,而Osmsh4突变体的单氨基酸替换正好发生在保守的MUTSd结构域上。我们通过转基因互补实验以及对OsMSH4基因TOS17突变体的调查证实OsMSH4就是目的基因。荧光定量、GUS染色和组织原位杂交分析表明,OsMSH4主要在生殖器官中表达,而且呈现一种动态变化,在减数分裂时期花药的性母细胞中表达最高,随后表达水平逐渐降低直至消失。亚细胞定位显示OsMSH4蛋白主要定位在细胞核中,与其功能相一致。4.前人研究表明MSH4蛋白能和MSH5蛋白一起互作形成异源二聚体结合到Holliday连接体上来稳定单链入侵,通过酵母双杂交和体外pull-down实验我们证明OsMSH4也能和OsMSH5互作,而且突变后的Osmsh4就丧失了与OsMSH5的互作能力;此外,我们还发现OsMSH5能够与OsRPA蛋白复合体中的OsRPA1a, OsRPA2b,OsRPA1c和OsRPA2c四个亚基互作。因为OsRPA复合体是结合到D环和单链DNA上的,说明在减数分裂过程中,OsMSH4/OsMSH5异源二聚体与OsRPA复合体互作来共同调控 Holliday 交叉(Holliday junction)第二链的捕获(second-end capture, SEC )。5.利用以亚洲栽培稻滇粳优1号(DJY)为受体,非洲栽培稻IRGC102295为供体构建的近等基因系(near-isogenic lines, NIL),我们鉴定了种间杂种花粉不育基因S39。细胞学观察发现杂种F1的花粉表现为半不育,属于染败,败育的花粉粒比正常的偏小且形状不规则,淀粉积累少,花粉粒壁发育不完全。进一步的研究发现F1中败育的花粉粒主要为DJY类型,败育时期发生在小孢子中期向晚期转变的过程,由于此过程的滞后导致DJY型的配子不能形成正常的三核花粉,淀粉积累也不完全。6.利用次级分离群体我们将S39定位在第12号染色体79-kb的区间,该区域一共预测了 13个候选基因。对该区间进行测序发现两亲本序列存在较大的差异,其中NIL比亲本DJY多了约82-kb的序列。进一步分析发现S39位于一个400-kb左右的品种间相互倒位区间。荧光定量结果显示定位区间几个基因在花药中高表达,然而单个基因的转基因实验却没有得到互补表型。遗传分析发现S39位点是一个普遍存在于种间和亚种间的杂种不育位点,而广亲和品种Dular,Kasalath在此位点携带有亲和基因。
[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.

【学位授予单位】：南京农业大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S511

【参考文献】