【作者】 李万昌；

【导师】 万建民；

【作者基本信息】 南京农业大学 ， 作物遗传育种， 2006， 博士

【摘要】 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发展方向。雄性不育突变体在植物的育种、遗传学、生殖生物学和分子生物学的研究中提供了很好的材料。为了更好地理解花粉发育的分子机制，需要识别和分离涉及此过程各个阶段的所有基因，并对其特性进行分析。有关水稻半不育的报道多以籼粳杂种半不育为主，并提出了很多假说，但半不育的分子机制仍不清楚。究其原因，这种半不育是由籼粳间互作引起的，遗传基础较为复杂，不同的组合可能具有不同的不育位点，研究起来较为困难，若能用稳定的半不育材料进行研究，阐明半不育产生的分子机制，对克服生产中遇到的半不育问题具有重要的借鉴意义。为此，本研究对稳定的半不育突变体材料W207-2进行了半不育机制的研究和花粉半不育基因的精细定位，所得结果为从分子水平上揭示水稻及其它禾本科作物半不育性的生物学基础具有重要的指导意义。本论文主要研究结论如下：1．用突变体材料W207-2分别与粳稻品种Nipponbare和广亲和品种Dular（籼稻）进行正反交分析表明：W207-2的半不育性受隐性核基因控制，不受细胞质效应的影响。在双目解剖镜下观察到W207-2的花药瘦瘪且开裂性差，而Nipponbare的花药饱满且开裂性好。由于花药开裂性差，造成花粉散出量减少，在荧光显微镜下观察到散落在柱头上的花粉粒和萌发的花粉粒总数减少，继而导致小穗育性降低；花粉的半不育性可能是造成花药开裂性差的原因之一。2．通过对W207-2／Nipponbare的F₂群体分析表明，半不育性受生育期影响较小，花粉育性与小穗育性呈极显著正相关（r=0.9119）；对W207-2/Nipponbare的F₂群体中291个单株和W207-2／／W207-2／Nipponbare群体中137个单株的遗传分析表明，W207-2的半不育性受1对隐性核基因控制；分别用W207-2和Nipponbare的花粉给W207-2及不育系六盐189A、丙8979A和9522A进行辅助授粉，验证了W207-2的半不育性主要表现为花粉半不育，雌配子育性正常。3．通过石蜡切片对Nipponbare和W207-2的小孢子发育过程进行观察，发现W207-2的花药在造孢细胞和花粉母细胞时期，药室中有染色极深的“染色质黑块”，造成花粉母细胞减数分裂异常而导致小孢子部分败育；W207-2的绒毡层延迟解体是其小孢子部分败育的另一原因。此外，W207-2的药隔微管束发育不良及药室发育异常也可能是其半不育性的一个原因。用整体染色-透明法观察Nipponbare和W207-2的胚囊育性，发现其育性正常。W207-2的半不育性主要是由其雄配子发育不良造成。4．利用间接酶联免疫（ELISA）检测技术，测定了水稻半不育突变体W207-2及其野生型Nipponbare在幼穗发育过程中叶片、幼穗和花药中内源IAA，GA₄，iPA，ZR和ABA含量的动态变化。结果表明：在W207-2的叶片，小穗和花药中IAA，GA₄和iPA的含量均低于Nipponbare，且IAA+GA₄+iPA与ABA+ZR之比值也始终低于Nipponbare，而ZR和ABA的含量则高于Nipponbare，提示W207-2叶片，幼穗和花药中IAA，GA₄，iPA，ZR和ABA含量出现了异常，且IAA，GA₄和iPA亏缺以及ZR和ABA盈积可能与半不育发生有关。5．选择花粉育性作为育性指标，随机选择W207-2／Dular F₂分离群体中的20株半不育单株及20株可育单株，分别将各株的叶片等量混合后提取DNA，构成半不育混合基因组池和可育混合基因组池。对两混合基因组池进行全基因组SSR分析，分别在第5、7和8染色体上检测到有多态的标记。随后用W207-2／Dular F₂群体中的182个单株构建了第5、7、8染色体的分子连锁遗传图谱，并进行了QTLs检测，在第8染色体上标记RS41和RM6356之间检测到1个花粉半不育位点，命名为pss1，其LOD值为48.3，贡献率为70.5％。6．以W207-2与Nipponbare杂交F₂群体中291个单株对花粉半不育基因进行定位，确定了原始花粉半不育基因突变位点为位于第8染色体上的pss1，即由正常品种Nipponbare变为花粉半不育W207-2是pss1位点上基因突变的结果。进一步用9600株W207-2／Dular F₂分离群体中2100株纯合隐性半不育个体将该半不育基因pss1精细定位于CAPs标记L2和dCAPs标记L3之间，距两标记的距离均为0.02cM。两标记位于同一PAC克隆P0470F10上，物理距离约为28kb，对此片段进行分析，预测到5个完整的开放阅读框，分别为：putative ribonuclease PH，putative avr9 elicitor response protein，kinesin1-like RNA-binding protein，putative RNA-binding protein RNP-D precursor和putative 40S ribosomal protein S13。更多还原

【Abstract】 Male-sterile mutations provide an ideal source material for a range of genetic andmolecular biological studies of reproductive biology. In order to well apprehend themolecular mechanism of pollen development, all genes involved in this coursedevelopmental stage must be identified and characteried.Many studies on the hybrid semi-sterility between indica and japonica have beenreported and many hypothesizes have been put forward, but the molecular mechanism ofsemi-sterility has been unknown. It is difficult to study this problem due to this hybridsemi-sterility causing by indica-japonica interaction. The hybrid genetic foundation iscomplex, so hybrid semi-sterility in different varietal combinations may be differentsterility loci. If we can use a stable genetic semi-sterile cultivar to study the semi-sterilemolecular mechanism, it would be a reference for overcoming the semi-sterility inpractices.W207-2 is a stable mutant, and mutants are favorable materials for studying geneexpression and function analysis, at the same time, analyzing mutants is also the develop-ment aspect for researching the functional genomes. The entire rice genome has beensequenced and publicly considered to be the genome of the model plant in grass, at thesame time, there is synteny between rice and other grass genome. Therefore, there is animportant use for reference to other grass plants through disclosing the biology basis inmolecular levels.The main results were as follows:1. On the basis of analysis with reciprocal F₁ hybrids between the mutant line W207-2and its original type Nipponbare（O. japonica） and a wide compatibility variety Dular（O.indica）, the semi-sterility was found to be controlled by recessive nuclear genes with nocytoplasmic effect. The anther of W207-2 was thinner and almost indehiscent, while that ofNipponbare was plumper and dehiscent. The anther indehiscence of W207-2 was found toreduce the numbers of pollen grains dropped onto stigma per spikelet. Thus the germinated pollen grains were reduced and induced the spikelet semi-sterility of W207-2. The pollensemi-sterility may be one of the causes that make the anther indehiscent.2. Analysis of the F₂ population of W207-2/Nipponbare suggested that the correlationbetween spikelet fertility and pollen fertility was significant （γ=0.9119） and the semi-sterility was independent of growth duration. Genetic analysis of 291 plants in W207-2/Nipponbare F₂ population and 137 plants in W207-2/W207-2/Nipponbare populationindicated that this semi-sterility was controlled by a recessive nuclear gene. W207-2 andsterile line Liuyan189A, Bing8979A, 9522A was hand-pollinated with the pollen ofW207-2 and Nipponbare, respectively, and the semi-sterility of W207-2 was found to bemainly caused by the pollen semi-sterility while the female gamete fertility was normal.3. The developmental process of microsporogenesis in anthers of Nipponbare andW207-2 were examined with light microscope. Some special abortive types were detectedin the sporogenous cells or microspore mother cells, and their structure was destroyed, theydeveloped into abnormal black clumps. The main reasons of pollen abortion of W207-2 are:（1） The meiosis of pollen mother cell in W207-2 was abnormally. （2） Tapetum cell delayeddisintegration. And the aberrant vascular bundle of connective tissue and abnormal theca ofanther was another cause of semi-sterility for W207-2. Whole-stain clearing method wasused for evaluating embryo sac fertility of Nipponbare and W207-2. The embryo sacfertility was normal, and the male gamete developmental abnormality of W207-2 was themain cause for the semi-sterility of W207-2.4. Contents of IAA, GA₄, iPA, ZR and ABA were measured by enzyme-linkedimmunosorbent assay （ELISA） in leaves, panicles and anthers of W207-2 and its originaltype Nipponbare during panicle development. IAA, GA₄ and iPA contents and the ratios ofIAA+GA₄+iPA to ABA+ZR in leaves, panicles and anthers of W207-2 were shown to belower than those of Nipponbare, while ZR and ABA contents in W207-2 higher than thosein Nipponbare. It is proposed that the changes in contents of these endogenesis hormone inW207-2 are abnormal, and IAA, GA₄ and iPA deficiency and ABA as well as ZRaccumulation might be related to the generation of semi-sterility in rice.5. Twenty semi-sterility and twenty fertility plants were random selected from the F₂population of W207-2/Dular according to pollen fertility, then genomic DNA from themwas pooled to make up the fertile and semi-sterile DNA bulks, respectively. A total of 565SSR markers throughout the rice genome were surveyed for their polymorphisms betweenthe fertile and semi-sterile bulks and between the two parents. Five SSR markers including RM178 on chromosome 5, RM118 on chromosome 7, RMS06, RM152 and RM6863 onchromosome 8, showed polymorphisms between the two bulks and between the two parents.Based on the linkage map of chromosomes 5, 7 and 8, which were constructed using 182random individuals from W207-2/Dular F₂ population, only one major QTL （pss1）controlling the pollen semi-sterility was identified with a LOD score of 48.3 and a PVE（phenotypic variation explained） of 70.5% in the region between markers RS41 andRM6356 on chromosome 8.6. The pss1 locus that located in the region between markers RS41 and RM6356 onchromosome 8 was confirmed by using another 291 individuals in F₂ population ofW207-2/Nipponbare. To fine-map pss1, 2100 recessive homozygous semi-sterile plantswere selected from a large W207-2/Dular F₂ population to identify the recombinationbetween the pss1 locus and the tightly linked molecular markers. Finally, pss1was mappedon the region between one CAPs L2 and one dCAPs L3 marker with the genetic distancesof 0.02 cM, respectively. Both L2 and L3 markers were located on a same PAC cloneP0470F10 with physical size of about 28 kb. Five open reading frames were predicted by asequence analysis of this fragment. They were as follows: putative ribonuclease PH,putative avr9 elicitor response protein, kinesin1-like RNA-binding protein, putativeRNA-binding protein RNP-D precursor and putative 40S ribosomal protein S13.更多还原