一个水稻灌浆突变体的遗传分析与基因定位

发布时间：2018-08-23 11:20

【摘要】：水稻是世界重要的粮食作物,也是植物基因组研究的模式植物。以收获籽粒为目的的水稻生产,其籽粒灌浆是生长发育过程中极为重要的阶段,涉及复杂的遗传调控网络和环境互作,直接关系到水稻产量和品质的形成。通过对水稻灌浆突变体的研究,挖掘控制水稻灌浆速率的新基因,对深入认识水稻灌浆机理,细化水稻灌浆过程具有重要意义；也能加强对水稻籽粒灌浆过程中糖类代谢途径生理因子和遗传因子的认知,更好地了解灌浆充实的内在信息,并能促进水稻高产优质品种的选育,达到高产优质高效的目的。本研究报道了宜香1B经EMS诱变而获得的一个水稻籽粒灌浆延长突变体gefl (grain extended filling 1),并主要对其进行了农艺性状调查、生理生化特性测定、组织细胞学观察、相关基因表达分析、遗传分析以及基因定位等方面的研究。主要结果如下：1.通过对突变体gef1与野生型宜香1B的主要农艺性状及稻米品质测定发现,与野生型相比,突变体株高无明显变化,但穗长缩短、结实率降低、每穗实粒数减少。在种子形态方面,突变体种子的粒长、粒宽和千粒重均增加,长宽比无明显变化,且进入灌浆期突变体籽粒颖壳外表面会产生不规则的深色斑点。在稻米品质方面,与野生型相比,突变体gef1的出糙率无明显变化,胶稠度、蛋白质含量与整精米率显著降低,垩白率和垩白度均增加,从而造成米粒透明度降低。2.通过组织细胞学观察发现,与野生型宜香1B相比,突变体gef1穗轴、倒一节间和倒二节间相同部位横切面的大维管束的平均个数减少,两者之间对应部位大维管束个数差异变化幅度为2至7个;穗轴壁和茎秆壁的厚度无明显变化,髓腔面积略为减小。颖壳内表皮电镜扫描结果表明,与野生型比较,突变体颖壳内表皮光滑,细胞细长且数目增多。淀粉体电镜扫描结果显示,突变体的淀粉体呈球形,排列松散；而野生型淀粉体呈无规则多边形,排列紧密。3.水稻籽粒灌浆速率分析发现,野生型宜香1B在花后0-18天处于快速灌浆期,大量的同化物被转运至颖果中,颖果干、鲜重持续增加；而突变体gef1的快速灌浆期维持有36天,并且其籽粒的灌浆速率明显低于野生型。水稻颖果发育动态分析表明,突变体gef1颖果前期发育缓慢,发育完全成形较野生型长约12天；突变体gef1从开花至种子成熟所经历的时间比野生型迟30天左右。4.生理指标测定发现,野生型宜香1B籽粒中的可溶性糖含量呈单峰曲线变化,且在整个发育过程中含量维持在较低水平；而突变体gef1籽粒中可溶性糖含量却呈现双峰趋势,且最大值明显大于野生型。野生型籽粒中可溶性糖含量在受精15天后开始下降,而突变体籽粒中可溶性糖含量在受精25天后才开始下降,并且在花后25天之前,籽粒中可溶性糖的积累效率与含量明显高于野生型。开花30天后,gef1籽粒中的可溶性糖含量又急剧增加,表明该时间段籽粒中可溶性糖的积累效率明显高于淀粉的合成效率。突变体颖果叶绿素含量在开花40天后才开始急速下降,说明颖果表皮持绿时间较长,背部维管束能维持长时间的运输功能。5.通过糖代谢相关基因的表达分析发现,相比野生型,SUS3在突变体gef1灌浆初期的变化幅度不明显,OsSSIIIa与SUS3类似,灌浆初期在gefl中的表达始终维持在某一较低水平,而在野生型中却逐渐升高。此外,OsAGPL2与OSAGPS2b在突变体与野生型灌浆初期的表达存在明显差异,在突变体中的表达均低于野生型,并且二者都呈现逐渐升高趋势。6.通过对gef1／宜香1B、gef1/02428和gefl/Kitaake杂交分析表明,三个组合的F1植株均表现正常,说明该突变性状受隐性基因控制。三个杂交组合F_2群体中突变表型单株数与正常表型单株数的分离比均符合3：1,表明该突变性状受一对单隐性核基因控制。7.以gef1/02428的F_2群体作为初定位群体,选取均匀分布于水稻12条染色体上的510对SSR标记进行亲本间多态性分析,并采用BAS(bulk segregation analysis)法对混池进行筛选。结果发现,在水稻第3染色体短臂的标记RM5474、RM7576.A3-7和A3-8在DNA混池间存在多态性,且与目的基因较连锁。为了进一步对目标基因进行定位分析,利用在该区间内寻找的SSR多态性标记以及根据粳稻日本晴和籼稻9311序列进一步设计的InDel标记,最终将该基因定位在InDel3-1与InDel3-2之间,物理距离约为198Kb。该区间共有21个开放阅读框,候选基因的进一步鉴定工作正在进行中。
[Abstract]:Rice is an important food crop in the world and a model plant for plant genome research. Grain filling is an extremely important stage in the process of rice growth and development for the purpose of harvesting grain. It involves complex genetic regulatory networks and environmental interactions, and directly affects the formation of rice yield and quality. The study of mutants and the discovery of new genes controlling rice grain filling rate are of great significance to understand rice grain filling mechanism and refine rice grain filling process; it can also strengthen the cognition of physiological and genetic factors of sugar metabolism pathway in rice grain filling process, better understand the inherent information of grain filling, and promote rice grain filling. In this study, a rice grain extended filling mutant Gefl (grain extended filling 1) was obtained from Yixiang 1B by EMS mutation, and its agronomic characters, physiological and biochemical characteristics, Histocytological observation and related gene expression were investigated. The main results were as follows: 1. The main agronomic traits and rice quality of mutant gef1 and wild type Yixiang 1B were determined. Compared with wild type, the mutant had no significant change in plant height, but shortened panicle length, reduced seed setting rate and reduced grain number per panicle. The grain length, grain width and 1000-grain weight of the mutant increased, but the ratio of length to width did not change significantly, and irregular dark spots appeared on the outer surface of the glume during grain filling. Compared with wild type Yixiang 1B, the average number of large vascular bundles in the same section between the first and second internodes of mutant gef1 decreased, and the difference of the number of large vascular bundles in the corresponding parts between them was 2-7. Compared with the wild type, the inner epidermis of the mutant glume was smooth, the cells were slender and the number of cells increased. The amyloplast of the mutant was spherical and loosely arranged, while the wild type was irregular. The analysis of grain filling rate showed that wild type Yixiang 1B was in rapid filling stage from 0 to 18 days after anthesis, a large number of assimilates were transported to caryopsis, caryopsis trunk and fresh weight continued to increase, while mutant gef1 maintained rapid filling period for 36 days, and its grain filling rate was significantly lower than that of wild type. Dynamics analysis showed that the mutant gef1 developed slowly at the early stage of Caryopsis and took about 12 days longer than the wild type, and the mutant gef1 experienced about 30 days later than the wild type. The content of soluble sugar in wild-type grains began to decrease after 15 days of fertilization, and the content of soluble sugar in mutant grains began to decrease after 25 days of fertilization, and then decreased after 25 days of anthesis. 30 days after flowering, the content of soluble sugar in gef1 grains increased sharply, indicating that the accumulation efficiency of soluble sugar in the grains was significantly higher than that of starch. The results showed that the expression of SUS3 in mutant gef1 was not significantly different from that in wild type. OsSSIIIa was similar to SUS3, and the expression of SUS3 in Gefl remained at a low level at the early filling stage. In addition, the expression of OsAGPL2 and OSAGPS2b in the mutant and wild type at the early filling stage was significantly different, and the expression of OsAGPL2 and OSAGPS2b in the mutant was lower than that in the wild type, and both of them showed a gradual increasing trend. 6. The results of gef1/Yixiang1B, gef1/02428 and gefl/Kitaake hybridization analysis showed that F1 of the three combinations was planted. The results showed that the mutant trait was controlled by recessive genes. The segregation ratios of single plant number of mutant phenotype and single plant number of normal phenotype in the three F_2 populations were all 3:1, indicating that the mutant trait was controlled by a pair of single recessive nuclear genes. 7. The F_2 population of gef1/02428 was selected as the initial population and evenly distributed in 12 rice. The results showed that the markers RM5474, RM7576.A3-7 and A3-8 on the short arm of chromosome 3 were polymorphic and linked to the target gene. Location analysis showed that the gene was located between InDel 3-1 and InDel 3-2 by using the SSR polymorphism markers searched in this region and further designed InDel markers based on the sequences of Japonica rice Nipponqing and Indica rice 9311. The physical distance was about 198Kb. There were 21 open reading frames in this region, and further identification of candidate genes was under way. Middle.
【学位授予单位】：四川农业大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：S511

【相似文献】