【作者】 王洋；

【导师】 万建民；

【作者基本信息】 南京农业大学 ， 遗传学， 2013， 博士

【摘要】 水稻是世界上重要的粮食作物之一,全球一半以上的人口,包括几乎整个东亚和东南亚的人口,都以稻米为食。在我国,水稻种植面积占全国粮食作物的1/4,并且产量占粮食作物的一半以上。可见水稻生产的好坏直接影响我国粮食供应和人民的生活水平。我国的人口还在持续的增长,随着城市建设的发展,土地的荒漠化,耕地面积的逐年减少以及其它农作物对耕地的需求所带来的竞争,要确保水稻的供给必须提高水稻的单产。而育性的高低直接影响着水稻的产量,可是在水稻育种过程中和水稻生产过程中,总是不可避免的会出现育性降低的现象。不同品种间组合带来的内部因素以及高温,低温,病害,等外部因素都影响水稻的育性突变体是研究基因表达与功能分析的良好试验材料,也是当前功能基因组学研究的热点之一,本研究从育性突变体入手,首先获得育性突变体材料,接着从细胞水平分析突变体育性降低的原因,从基因水平研究其分子机理。结果,我们从实验室之前得到的有一个Q6537突变体中分离到了一个新的水稻育性基因,对这个基因和实验室已经克隆的花粉半不育基因(PSS1),进行了深入研究。这些结果丰富了人们对水稻育性的认识和理解。本论文的主要研究结果如下：一.水稻育性突变体的筛选从10000份T-DNA插入突变体中,最终筛选到了20个稳定遗传的低育性突变体和7个同时伴随其它性状变异的突变体,遗传分析表明它们受单基因的控制。对其中的一个颖花突变体M3进行了定位,结果表明与水稻extra glume1基因等位。二.水稻三体不育突变体Q6537的遗传分析及OsMSH4的克隆和功能分析1.本实验室之前获得了一个水稻雌雄不育突变体Q6537,这个突变体来源于水稻的四倍体花药培养。Q6537的自交后代可以稳定的产生结实率为0%的Q6537M,结实率为29.2%的Q6537和少数其它类型,前面两种类型的比例约为3：1。2.从34个Q6537株系共1360个单株中,得到19株正常结实的植株,14株出现育性的分离,分离比为3：1(218株可育株Q6537WT比65株不育株Q6537M)。3.通过对Q6537的减数分裂观察,发现Q6537的中期Ⅰ会出现12个二价体+1个单价体或11个二价体+1个三价体的染色体构型,这表明Q6537是一个三体水稻。4.利用Q6537不育突变体与籼稻9311配制组合,筛选其中的二体F1群体,用做基因的定位群体。利用分子标记的连锁,将定位区间缩小至60kb的范围,通过测序发现区间内OsMSH4的保守位点发生了一个单碱基的突变。5.通过对Q6537M的减数分裂观察,发现大部分的同源染色体间不能正常形成二价体,导致同源染色体的分离出现紊乱,雄配子无法获得12条整套染色体,碘-碘化钾染色表明花粉完全不育。6.水稻中的()sMSH4与拟南芥中的基因AtMSH4同源,这个基因参与同源染色体的交换,AtMSH4基因的突变使拟南芥的减数分裂时期出现大量单价体,育性降低。7.基因的定量PCR验证OsMSH4是减数分裂特异表达的基因,同时,OsMSH4基因的突变对其它减数分裂基因的表达量没有明显的影响。三.水稻半不育突变体W207-2的基因PSS1的功能分析以及与其互作的基因的筛选本实验室先前从稳定的水稻半不育突变体W207-2中克隆了一个花粉半不育基因PSS1, PSS1编码的是一个驱动蛋白,W207-2中PSS1的单个氨基酸的改变使蛋白的功能丧失,造成部分的减数分裂细胞出现少量的单价体,使花粉出现半不育的表型,导致花药发育成熟后花粉所提供的膨压不足,影响花药的正常开裂,从而使W207-2出现半不育的表型,关于这部分的详细工作已经发表。下面是后续的实验主要包括：1.水稻和拟南芥的亚细胞定位结果表明PSS1主要定位在细胞核中,这在空间上与减数分裂染色体和纺锤体出现的位置相一致。2.体外的微管结合实验表明,单个氨基酸的突变使PSS1蛋白丧失了与微管结合的能力。3.在pssl突变体中,没有观察到纺锤体出现明显的缺陷,仍然以染色体出现单价体为主要缺陷,单价体的形成可能是染色体配对异常导致的或者是由染色体的运动异常导致的,目前的论断更倾向于后者。4.通过转RNA干扰载体,对PSS1基因的表达进行干扰,使转基因株中PSS1的表达量明显的降低,结果产生了与W207-2一致的表型。5.利用酵母双杂交的方法,通过对水稻的穗cDNA文库进行筛选得到两个与PSS1互作的蛋白,分别是谷氨酸合酶Glutamine synthetase(GS)和Targeting protein for Xklp2(TPX2)。更多还原

【Abstract】 Rice is one of the most important food crops in the world, more than half of the global population, including almost the entire population of east and southeast Asia, are feed on rice. In China, rice planting area accounting for1/4of food crops’s, and yield for more than half of food crops. The production of rice directly affect Chinese food supply and people’s living standard. China’s population continues to increase, with the development of city construction, land desertification, arable land decrease year by year and the cultivated land demand of other crops, to ensure the supply of rice must increase rice yield per unit. And sterility directly affects the production of rice, but in the process of rice breeding and rice production, always inevitably experience decreased fertility. Combination between different varieties as internal factors as well as high temperature, low temperature, diseases, and so on of many external factors will be more or less affect the sterility of rice, which makes the fertility changes of rice is extremely complex and appears to be no rules to follow.Mutants is the ideal source material for studying gene expression and function, it is one of the current hot topics in the study of functional genomics. In this study, first we get sterility mutant materials, then analysis the fertility reduced reason at cytology level, finally we get the genes function to explain the molecular mechanism. As a result, we isolated to a new rice sterility gene from Q6537mutants and study the pollen sterility gene (PSS1)which have been cloned. The results enrich our understanding of rice sterility. Main research results of this paper were as follows:(A) rice sterility mutants screeningFrom about10,000T-DNA insertion mutants, that we picked up the20stable genetic lines with low fertility that separated by3:1ratio (controlled by a single gene) and7lines in addition to alone with other phenotype variation. On one of these mutants, M3has carried on the map based cloning, results show that it was allelic of rice Extra glume1.(B) genetic analysis of rice trisome sterility mutant line Q6537,from this line we cloned OsMSH4and analysis the gene’s function1. Our lab have been got a rice male sterile mutants Q6537, the mutant was collected from an anther culture mutants population originated from autotetraploid indica/japonica hybrid H3774(H2088×H891). we surveyed419progenies’individual from Q6537and found the phenotypic ratio of Q6537to Q6537M is111to308(roughly1:3)2. We then attempted to obtain the wild type of mutant Q6537M from the progeny of Q6537. As a result,19normal seed setting plants were selected out of1360individuals consisting of34Q6537plant lines. Among their progeny,14of them displayed further fertility segregation fitting to a ratio of3:13. We then surveyed the meiotic cell to inspect chromosome constitution of Q6537. At metaphase I, other than a full set of12pairs chromosome, one extra chromosome presenting either outside the equatorial plate, or, in most situation (over90%of total56spreads), synapsed with a bivalent to form trivalent, we deduced that Q6537is a typical trisomic line, trisomic Q6537make the inheritance accessible of a full sterility mutant Q6537M.4. To map the gene, we crossed the trisomic Q6537as female parent with9311, an Indica variety, to construct a genetically segregating population. Using the molecular markers, further mapped into a60-kb interval, the mapped region contains9recognizable open reading frames (ORFs). One of the ORFs (ORF8) encodes a protein homologous to AtMSH4, in which there is a single nucleotide substitution at codon482(G/C) in the third exon, resulting in an amino acid change from Ala-118to Pro.5. Through the Q6537M meiotic observation, the most obvious defects became apparent at diakinesis when all meiocytes (>200cells) had<12bivalents, mostly at the range of2-4. The remaining are unsynapsed univalents. Subsequently all bivalents and some univalents aggregated on the metaphase I equatorial plane, make the pollens of Q6537M fully empty that stained by iodium potassium iodide.6. OsMSH4is the homologous of AtMSH4gene in arabidopsis, the gene involved in the exchange of homologous chromosomes, the mutations of Atmsh4gene in the arabidopsis make a large number of univalent during meiosis period, it also make the fertility reduced significant but still have some fertility.7. Gene’s quantitative PCR indicate OsMSH4was meiosis specific gene, at the same time, there are no obvious different expression between Q6537M and WT with other meiotic gene at the upstream and downstream of OsMSH4.(C) Analysis the function of PSS1gene that from a semi-sterility mutant line W207-2and found other proteins that interact with PSS1Our laboratory previously got a stable heredity rice semi-sterility line W207-2, and cloned pollen sterility gene PSS1from the line, PSS1is a kinesin, there is a single amino acid change of the protein which make it lose its function, causing part of the meiotic cell appear a small amount of the univalents, makes the pollen exhibit infertile phenotypes, lead to reduced mechanical pressure generated in the pollen sacs, affect the normal anther dehiscence, allowing the semi-sterility phenotype in W207-2, detailed work has been published about this part. Here are the follow-up experiments:1. PSS1-GFP subcellular localization in rice and arabidopsis protoplast cells showed that it mainly locate near and in the nucleus space, where chromosomes and spindles appeared at meiosis stage2. Microtubules combined experiments in vitro show that a single amino acid mutation makes the PSS1protein lost the ability to combine with microtubules.3.In the pssl mutants, no spindle appear observed flaws. The univalents are the main defects, the formation of the univalents may be the result of abnormal chromosome pairing or is caused by abnormal chromosome movement.4. By the RNA interference of the PSS1gene expression, it make the expression capacity of PSS1in the transgenic plant is significantly lower, the phenotypes of the transgenic plants are the similar with the phenotypes of W207-2.5. By using yeasts two-hybrid method, screening with rice panicle cDNA library, we found two proteins are glutamate synthase Glutamine synthetase (GS) and Targeting protein for Xklp2(TPX2) could interact with PSS1.更多还原