【作者】 初明光；

【导师】 李平；

【作者基本信息】 四川农业大学 ， 作物遗传育种， 2009， 博士

【摘要】 雄性不育是开花植物一种较为常见的生殖障碍现象,主要表现在有性繁殖过程中不能产生正常可育的雄配子体,但雌性器官发育正常。它是植物为适应外界环境,经过长期进化的结果。作为单子叶植物的模式植物,水稻是研究植物生殖发育机理的一个良好材料。水稻花粉发育过程主要体现为花粉囊中的小孢子母细胞经过减数分裂产生小孢子,后者经液泡化和有丝分裂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。从雄蕊发育到成熟花粉的释放等任何一个环节发生了基因突变,均能引起花粉发育异常,最终导致雄性不育。近年来,随着水稻基因组测序的完成、EST库和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。XS1是一个源于自然突变的水稻雄性不育突变体,自然状况下结实率低,套袋自交则表现完全不育。经过多代自交分离,确认该突变性状能够稳定遗传。与野生型比较,该突变体花丝细长,花药干瘪,呈白色或者淡黄色,在生长后期叶片仍呈深绿色。碘染证实,XS1花药壁内没有花粉粒着色,属于典型的“无花粉型不育”类型。石蜡切片发现,在花粉母细胞开始发育到减数分裂完成这一期间,突变体和野生型相比较无显著差异,小孢子的形成过程是完全正常的,花药的三层壁的变化与发育也与野生型一致。在初生造孢细胞形成的同时形成三层壁;在四分体时期绒毡层胞质呈浓缩状态,颜色加深;到了小孢子时期绒毡层降解成山丘状,中层细胞变得狭窄;液泡化后期,中层和绒毡层的原生质体降解消失。四分体末期形成小孢子后,突变体的小孢子发育开始出现异常。野生型小孢子后期顺利进行液泡化,体积逐渐增大,核发生浓缩,最后经有丝分裂形成正常的花粉粒。而突变体小孢子后期虽有进行液泡化的趋势,但是在液泡化早期即出现小孢子粘连,继而形成凝聚物,未能进行有效的液泡化,后期凝聚物逐渐降解形成空腔。因此,突变体花粉发育障碍主要表现为小孢子液泡化进程受阻,直接结果是花粉囊内无花粉粒形成,从而最终导致不育。以突变体作为母本,与多个育性正常的水稻材料杂交衍生F₂群体,或利用杂交F₁与突变体回交衍生BC₁F₁代群体,调查突变性状在各种背景下的分离。结果表明,所有群体的F₁植株均表现正常可育,F₂或BC₁F₁出现育性分离,说明该育性基因受核基因控制而不受胞质基因组的影响,可育相对不育为显性。在所有F₂群体中,育性分离均符合3:1的分离比例,而BC₁F₁植株则符合1:1的分离规律,表明该不育性状为单个基因控制的隐性突变。选择XS1/G630来源的F₂作为基因初步定为的群体,群体总数约为1800株,其中雄性不育单株为432株。以本实验室保存的、均匀分布于水稻12条染色体上的微卫星引物对亲本的SSR基因型进行检测,发现有103对引物在两亲本间存在多态性差异,对能揭示G630和XS1差异的标记先在小群体中（由2个亲本、来源于F₂的4株可育株和6株不育株构成）进行初步的连锁分析,选取在小群体中与基因表现连锁的标记,对F₂群体的所有隐性单株进行基因型扫描。结果发现,标记RM470、RM303、RM317、RM17411、RM5030、RM6748和RM17473与突变基因表现明显连锁。其中,RM470、RM303、RM317和RM17411位于基因同一侧,遗传距离分别为3.4、2.7、2.4和0.7 cM;标记RM5030、RM6748和RM17473位于基因另一侧,遗传距离分别为1.5、2.7和3.0 cM。据此,基因被定位在第4染色体微卫星标记RM17411和RM5030之间,两标记间的遗传距离为2.2 cM,同时,区间内的标记RM17414和RM3276与基因表现共分离。为了进一步缩小vrl所在范围,采用两个扩大的定位群体对目标基因进行定位。其中,群体Ⅰ（XS1/G630）总株数约有5260株,其中雄性不育株为1290株;群体Ⅱ（XS1/M63）总株数约为14360株,其中雄性不育株3540株。在初步定位的基础上,首先利用标记RM17411和RM5030之间公布的SSR引物分析亲本多态性,同时,利用水稻基因组序列信息开发新的SSR标记和InDel标记,进行“染色体步移”。结果发现,SSR标记FS15与vrl存在紧密连锁关系,遗传距离为0.16 cM;另外4个InDel标记D2-7、FID30、D8和FC4-2也与vrl紧密连锁,其中D2-7、FID30和D8与vrl的遗传距离分别为0.18、0.11和0.19 cM,标记FC4-2与vrl表现共分离。综合两个群体中定位的结果:RM17414、RM17434、RM7208、RM17438、D2-7和FID30位于基因vrl的同一侧,遗传距离分别为0.62、0.46、0.42、0.29、0.18和0.11 cM;而标记FS15、D8、RM3217、RM5503、RM17450和RM3276位于基因的另一侧,遗传距离分别为0.16、0.19、0.45、0.63、0.84和1.38 cM,同时,区间内标记FC4-2与基因共分离。FID30和FS15是基因两侧最近的标记,分别位于两个相邻的克隆上（AL607004和AL606683）,根据日本晴基因组数据,这两个克隆之间没有Gap,两个标记之间的物理距离约为48kb。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FID30和FS15之间（日本晴基因组数据）预测到8个Loci,分别是LOC_Os04g51080、LOC_Os04g51090、LOC_Os04g51100、LOC_Os04g51110、LOC_Os04g51120、LOC_Os04g51130、LOC_Os04g51140和LOC_Os04g51150。同时利用水稻全长CDNA文库、GO功能分类和SWISS PROTEIN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8个候选基因中,LOC_Os04g51130编码一种表达蛋白,未能给出预测功能;LOC_Os04g51150基因编码一种转座蛋白;对于其余6个候选基因预测到了可能的功能,分别可能与爬行酶、tRNA剪接内切酶、COBW domain蛋白、WD重复蛋白、ENTH（The epsin NH2-terminal homology）域和E2F相关蛋白有关。更多还原

【Abstract】 XS1 was derived from a spontaneous mutation and confirmed to be a no-pollen type mutant of male sterility in rice.The floret of the mutant,consisting of six stamens and one pistil,looks the same as that of the wild type in the male-female organs,except that the filaments are long and thin,and the anthers are withered in white transparence.It is confirmed that XS1 is a none-pollen-type mutant of male sterility for no pollen grains can be stained with I₂-KI solution and the anther locules are always hollow.Anther transverse sections indicate that the mutant microspores are abnormally condensed and agglomerated to form a deep-stained cluster at the late microspore stage,which results in the ceasing of the vacuolation process of microspores,and,therefore,the mutant forms no functional pollens for reproduce.During the heading stage,all the individual plants in the F₁ and F₂ progenies from the crosses between XS1 and other normal rice lines were investigated.In the four F₁ progenies,all plants exhibited wild-type phenotype,suggesting that the mutant trait is recessive.In the four F₂ populations,all the segregation rates of fertility and sterility plants fit the ratio of 3:1,and,additionally,in the 3 BC₁F₁ populations,all the segregation rates of fertility and sterility plants fit the ratio of 1:1.Genetic analysis processed in 4 F₂ populations and 3 BC₁F₁ populations reveal that the mutation is controlled by a single recessive gene,and it is termed as vrl（Vacuolation retardation l）.The polymorphisms between XS1 and other rice lines G630 and M63 were examined with 512 SSR markers and the most polymorphism richment population XS1/G630 was selected for mapping.Firstly,total 103 SSR polymorphic markers were selected and used to survey in a small populations which was composed of the two parents,four of wild type F₂ plants,and six of F₂ mutants plants.The result showed that 3 SSR markers RM470,RM303 and RM317 located on chromosome 4 were obviously associated with the XS1 phenotype.Then,the three markers were used to survey all the mutant plants in the same F₂ population,and were all verified to be linked to vrl with the genetic distance of 3.4,2.6 and 2.4 cM,respectively.In order to fine-map the vrl gene,two large segregation populations were generated from crosses of XS1/G630 and XS1/M63,respectively.Besides,sevaral SSRs,InDels markers were newly developed according to the publicly available rice genomic sequences.Reconbination analysis indicated that the vrl gene was finally located within a genetic interval of 0.27 cM,flanked by markers FID30,FS15,and co-segregated with marker FC 4-2.The delimitation region,according to the japonica rice genomic data,was estimated to be a 48 kb physical interval.Within the 48 kb physical interval,totally 8 putative genes were predicted by some sofiwares of the bioinformatics.LOC_Os04g51130 is a expressed protein with unknown function;LOC_Os04g51080 is a scramblase protein;LOC_Os04g51090 is a tRNA-splicting endonuclease positive effector-related protein;LOC_Os04g51100 is a putative COBW domain containing protein;LOC_Os04g51110 is a putative WD repeat-containing protein;LOC_Os04g51120 is an ENTH domain containing protein; LOC_Os04g51140 is an E2F-related protein;and LOC_Os04g51150 is a putative unclassified transposon protein.更多还原