【作者】 丁磊；

【导师】 李平；

【作者基本信息】 四川农业大学 ， 生物化学与分子生物学， 2009， 硕士

【摘要】 植物不育现象广泛存在于开花植物中,其主要表现是在有性繁殖过程中不能产生正常可育配子体。这种现象对植物本身的生长发育是不利因素,但是对于植物育性机理研究来讲却是一个良好的切入点。水稻是世界上最重要的粮食作物之一,其育性水平直接关系到产量的高低。同时,水稻作为单子叶植物的模式植物,其育性机理对于其他单子叶植物具有重要借鉴意义。水稻花粉发育由花粉囊中的小孢子母细胞经过减数分裂产生小孢子,然后进一步发育形成花粉粒,当花粉囊裂开时,花粉粒被释放出来并参与受精过程。水稻胚囊发育则是由胚囊母细胞经过减数分裂形成四分体,近合点端的形成胚囊,然后连续发生三次有丝分裂,最终形成七个细胞八个核的成熟胚囊。在雌雄蕊发育的一系列过程中,任何相关的基因发生变异,都会导致不育的发生。近年来,随着水稻基因组测序的完成、EST库、突变体库的构建及基因表达谱分析等工作的开展,对水稻不育的分子机理研究取得了很大的进展。在前期的研究中,我们从中花9号的转基因T₂代株系中发现一个表现雌雄均不育的突变体MFS-Z9,随后连锁分析证明其不育并非是由外源T-DNA的插入所引起。因此,本文对MFS-Z9进行了形态学、细胞学和遗传学及基因定位的研究,以期为该基因的图位克隆奠定基础。主要结果如下:1、突变体花丝细长,花药干瘪,呈白色或者淡黄色水浸状,叶片呈深绿色。以该突变体与其他育性正常水稻材料进行大量正反杂交均未获得杂交种子,说明该突变体为雌雄双不育突变体。给突变体授粉后发现,其子房有假膨大现象,膨大的子房内充盈着水,没有其他内含物。碘染证实MFS-Z9花粉囊为空腔,没有花粉粒着色,属于无花粉型败育,败育彻底。2、石蜡切片发现导致其雄性不育的原因主要在于小孢子母细胞在减数分裂时期降解和绒毡层细胞降解延迟。对减数分裂时期做了更细致的分期,发现在减数分裂小孢子母细胞时期,其发育与正常株无明显的差别,但是到了减数分裂细线期,小孢子母细胞开始降解,到二分体时期小孢子母细胞绝大多数几乎完全降解,而其绒毡层细胞则延迟降解并一直保留到成熟花粉时期。3、胚囊连续切片发现约95%胚囊为无内部结构分化,或约5%胚囊内部结构分化不彻底,不能形成有功能的8细胞胚囊。因此,该突变体雌雄配子的发育都不正常,从而表现出雌雄不育。4、由于该突变体高度不育,因此采用杂合体（ZHS）形式保存突变基因。利用ZHS与正常水稻材料如G630、kitake、Nippbare等杂交衍生含有突变基因的F₁、F₂（群体构建方法和鉴定过程详见“材料和方法”部分）。育性观察表明,所有F₁植株表现育性正常,而F₂出现育性分离,正常株与突变株的比例均符合3:1的分离比例,说明不育性状为一对基因控制的隐性突变,结合育性产生的细胞学原因,将该突变基因命名为MDMFS1（Multiplex Defective Male-femaleSterility）。5、利用512对SSR引物分析突变体和G630,筛选两个亲本间具有多态性差异的引物。差异标记分别在由亲本、4个可育株、6个不育株构成的小群体中进行初步的连锁分析,发现位于第2染色体上的SSR标记RM555、RM3732、RM3497和RM7215与突变性状存在连锁关系,然后用这4个标记对F₂所有的隐性单株（226株）进行扫描,证明这4个标记与突变性状表现连锁,而且RM555和RM3732位于基因同一侧,其遗传距离分别为1.1 cM和0.44cM,RM3497和RM7215位于基因另一侧,其遗传距离分别为0.44cM和0.88 cM。采用同样的方法在Kitake群体进行进一步定位,发现RM3865和RM12585与突变性状存在连锁关系,且与RM555和RM3732位于同侧,其遗传距离分别为0.36cM和0.09cM。6、利用网上公布的籼、粳2套基因组序列在定位区间开发SSR和Indel标记用于精细定位。差异分析显示新开发的SSR标记D051和Indel标记DI014、DI016和DI017在G630和突变体间表现出多态性,F₂群体连锁分析发现这些标记均与突变性状紧密连锁,其中D051与RM3497和RM7215均位于基因同一侧,与目标基因的遗传距离为0.22 cM。而DO014、DI016和DI017未检测到交换株,与突变性状表现出共分离。因此,突变基因被定位到RM12585和D051之间,其间的物理距离为99kb。7、采用TIGR Rice Browse、GRAMENE提供的在线预测软件,结合水稻相关的数据库对精细定位区域进行ORF预测,在标记RM12585和D051之间（日本晴基因组数据）预测到11个Loci,分别是LOC_Os02g08430、LOC_Os02g08440、LOC_Os02g08450、LOC_Os02g08460,LOC_Os02g08470,LOC_Os02g08480、LOC_Os02g08490、LOC_Os02g08500、LOC_Os02g08510、LOC_Os02g08520和LOC_Os02g08530。同时利用水稻全长CDNA文库、GO功能分类和SWISSPROTEIN蛋白数据库等对预测基因进行初步功能分析。结果显示,在这11个候选基因中,LOC_Os02g08430和LOC_Os02g08460推测编码的是未知蛋白,功能未知;LOC_Os02g08470推测编码的是保守未知蛋白,其功能也未知:LOC_Os02g08440编码具有WRKY和锌指结构的水稻TFs表达蛋白:LOC_Os02g08450推测编码转座蛋白,与转座有关;LOC_Os02g08480编码泛素降解蛋白;LOC_Os02g08490编码分子伴侣蛋白clpB 1;LOC_Os02g08500编码双组分调控蛋白:LOC_Os02g₀8510编码ZOS2-04-C₂H₂锌指蛋白;LOC_Os02g₀8520基因编码蛋白酶体T₁家族蛋白。LOC_Os02g₀8530基因编码蛋白激酶。更多还原

【Abstract】 Rice（Oryza sativa） is one of the most important food crops in the word.The rice genome sequence has been determined and a large number of mutants are available. Meanwhile,because of its smaller genome size（430Mb）,so rice is an excellent model plant for cereal developmental biology.Male and female sterility phenomenon occasionally occurring in plant,which is bad for the plant themselves but it offer a good chance for our understanding the mechanism of plant development.In rice anther development process,the microspore mother cell firstly undergoes meiosis to produce the microspore in the anther locule,and the microspore further developed into pollens through mitosis,then the anther splited and released maturity pollens for fertilization. Any factors participated abnormally in anther development would resulted in anther abnormality,which finally caused male sterility.The megaspore mother cell undergoes meiosis to form four haploid megaspores.The three of spores undergo programmed cell death and a chalazal spore becomes a functional megaspore.The megaspore then undergoes three sequential mitotic nuclear divisions,to generate the eight nuclei of the mature embryo sac.In the mature embryo sac,the micropylar end of the ovule has the egg cell and two supporting cells called synergids,while the chalazal end of the ovule has three cells of undetermined function called the antipodals that disintegrate prior to fertilization.In recent years,with the development of the rice genome sequencing and annotation,the molecular mechanisms of pollen development in rice are gradually clear.We reported the male and female sterile mutant,MFS-Z9,was generated from a T2 generation transgenic line of normal Japonica rice,Zhonghua 9.The sterility phenotype was subsequently confirmed not be the causation of the foreign T-DNA insertion by linkage analysis.The results of the mendelian genetics,genetic effect, morphological cytology and chromosome localization were as follows:1.With normal flowering,the mutant set no seeds when matured.Also no seeds are obtained neither the mutant is crossed as a pollen receiver nor as a pollen donor. The floret of the mutant consisting of 6 stamens and 1 pistil,looks the same as that of the wild type except that the filaments are long and thin and the anthers are withered in white transparence.The mutant could not produce any fertile seeds except some fake-enlarged ovules when it was crossed with several normal rice lines as pollen receivers.It is verified that in MFS-Z9 no pollen grains can be stained with 1%I₂-KI solution and the anther locules are always hollow.2.Anther transverse section indicates that microspores abnormally degenerated at the stage of meiotic,while the tapetum layer couldn’t normally degenerate and remained all the time.To gain a more detailed understanding of the abnormalities of the sporogenous and the tapetal cell,the meiosis stage was divided into several substages for observations according to the state of synapsis and condensation.No obvious differences were observed between the mutant and the wild-type rice during the early premeiosis stage.Morphological differences began at the stage of leptotene in the anther locules.During this stage,the microspores began to degenerate and the microspores were almost completely collapsed at the tetrad stage,while the tapetum layer couldn’t normally degenerate and remained all the time.3.In addition,more than 95%of the mutant embryo sac did not undergo differentiation and lacked visible nucellus cells in the ovule,and almost 5%of the embryo sac failed to develop into functional embryo sac with eight cells.Thus,mfs-Z9 displayed multiplex defects in the process of male-female organ development.4.Due to its serious sterility in both male and female organs,the mutation was maintained by heterozygotes（+/-,designated as ZHS,in Zhonghua 9 background）. ZHS（+/-） was first crossed with normal rice lines（+/+）,then all the generated F₁ plants（+/+ or +/-） were harvested separately,and approximately 100 seeds from each F₁ plant were sown to generate small F₂ populations to investigate the segregation of mutant phenotype.The segregation exactly indicated that the corresponding F₁ plant was in a heterozygote genotype（+/-）.All seeds harvested from the selected heterozygote genotypic F₁ plants（+/-） were then sown to produce large F₂ populations for further genetic analysis and mapping.According to this protocol,3 F₂ populations were generated from the cross of ZHS/G630,ZHS/Nipponbare and ZHS/Kitake, respectively.After flowering,all individual plants were subjected to fertility investigations and data were recorded.All F₁ plants exhibited the wild type phenotype, suggesting that the mutant trait was recessive.In the F₂ populations,the segregation of fertile plants and sterile plants fit a ratio of 3:1(X²＜X²_0.05,1=3.84),which indicated that the mutant phenotype was controlled by a single recessive nuclear gene. Considering the mutant phenotype and its defectives,the mutant gene was termed Multiplex Defective Male-female Sterility 1（MDMFS1）.5.Polymorphisms between MFS-Z9 and other rice lines,G630,Kitake and Nipponbare were examined,respectively,with 512 pairs of SSR primers.The polymorphic markers were subsequently used to survey in a small population including the 2 parents,4 wild type F₂ plants and 6 mutant F₂ plants.Results showed that 4 SSR markers RM555,RM3732,RM3497 and RM7215,located on chromosome 2,were obviously associated to the MFS-Z9 phenotype.Those 4 markers were then utilized to survey all 226 mutant plants which come from the ZHS×G630 in the same F₂ population.Thus,RM555 and RM3732 were verified to be linked to MDMFS1 on one side,with genetic distances of 1.11 and 0.44 cM,respectively,and the other two markers RM3497,RM7215 were verified to be linked to MDMFS1 on the other side, with genetic distances of 0.44 and 0.89 cM,respectively.The same approach for linkage analysis was employed in another population,ZHS×Kitake which contains 556 mutant plants.Another 2 polymorphic markers,RM3865 and RM12585,with 4 and 1 recombinants revealed,were confirmed to be linked to the MFS-Z9 phenotype,with genetic distances of 0.36 and 0.09 cM on the same side with RM555 and RM3732, respectively.6.To further narrow down the genomic region containing the MDMFS1 locus, more SSRs and InDels（Insertions and Deletions） markers between RM12585 and RM3497 were developed.Out of 80 newly developed markers,4 makers designated as DI014,DI016,DI017 and D051 were polymorphic in the population of G630×ZHS. Linkage analysis showed that marker D051 was tightly linked to MDMFS1 with genetic distance of 0.22 cM,and marker DI014,DI016 and DI017 were all co-segregated with MDMFS1.These results,together with the rice genome sequence data,suggested that the MDMFS1 locus was located on a 99-kb genomic fragment bordered by marker RM12585 and D051 on a single BAC clone on chromosome 2.7.In the 99-kb genomic interval of Nipponbare genome,totally 11 putative genes were predicted by TIGR RICE（http://rice.plantbiology.msu.edu/）.Out of them 2 genes are hypothetical protein（LOC_Os02g08430 and LOC_Os02g08460）,1 gene is conserved hypothetical protein（LOC_Os02g08470） and 1 gene is transposon protein （LOC_Os02g08450）;while the remaining 7 genes are expressed proteins. LOC_Os02g08440 is a member of the Superfamily of rice TFs having WRKY and zinc finger domains;LOC_Os02g08480 is ubiquitin fusion degradation protein 1; LOC_Os02g08490 is chaperone clpB 1;LOC_Os02g08500 is the two-component response regulator ARR12;LOC_Os02g08510 is zinc finger protein 1; LOC_Os02g08520 is proteasome subunit beta type 3 and the LOC_Os02g08530 is a phytosulfokine receptor precursor.更多还原