水稻籽粒中调控淀粉合成关键基因FLO12的图位克隆与初步功能鉴定

发布时间：2018-10-15 08:37

【摘要】：淀粉是水稻(Oryza satiza L.)种子中主要的贮藏物质,淀粉的含量和理化性质影响稻米的各项品质指标,同时水稻淀粉的积累水平还影响稻米产量。因此阐明水稻这一主要粮食作物的淀粉合成途径中的关键基因和调控网络具有重要理论意义与应用价值。本研究以采用MNU诱变获得的滇粳优1号籽粒粉质突变体flo12为研究材料,对其表型进行分析,进一步通过图位克隆的方法获得目标基因FLO12。主要结果如下:1.flo12种子去壳后,胚乳外观呈现粉质不透明,横截面观察显示主要是胚乳内部粉质。扫描电镜观察显示,野生型滇粳优1号的淀粉颗粒呈多面体,排列紧密,大小均一;突变体flo12淀粉颗粒呈规则圆形,排列松散,大小不一,颗粒之间存在较大间隙。与野生型相比,flo12的千粒重和粒宽极显著下降。2.发育中期的胚乳半薄切片观察显示,突变体flo12造粉体结构明显比野生型大,并且存在许多小的、零散分布的单粒淀粉颗粒。3.生理生化指标测定结果表明,与野生型相比,flo12总淀粉含量降低,直链淀粉含量显著下降,脂质含量极显著升高,同时flo12突变体中淀粉的尿素溶解特性与野生型相比也发生改变。4.利用图位克隆的方法获得FLO12。配制了flo12突变体与籼稻品种IR36的杂交组合,利用F2群体将突变基因定位在第四染色体长臂标记LJ4-28和LJ4-33之间的93 kb区域内。基因测序表明,该区间内基因Os04g0553000的第890个碱基,由C突变为T,导致其编码的第297个氨基酸由Thr(苏氨酸)突变为Ile(异亮氨酸)。5.构建互补载体转入flo12突变体,在T0代植株上同时收获到具有突变表型的粉质胚乳的种子和恢复表型的透明胚乳的种子,且透明种子与粉质种子的比例符合3:1分离比。因此,基因Os04g0553000就是我们寻找的控制突变体粉质胚乳表型的基因。6.对FLO12的时空表达进行分析发现,FL012基因为组成型表达模式,在叶片中表达量最高;其中在胚乳发育过程中,FLO12表达量也有所变化,在花后12天表达量最高。7.预测发现FLO12蛋白N端含有一个质体定位的信号肽。为了验证这个预测结果,在水稻原生质体中进行亚细胞定位。结果表明,FLO12-GFP的荧光信号能够与叶绿体自发荧光共定位,说明FLO12定位于质体中。8.对淀粉合成相关蛋白的表达进行分析发现,这些蛋白在野生型与突变体中的表达均无明显差异,说明FLO12的突变并不影响这些淀粉合成相关蛋白的表达。
[Abstract]:Starch is Rice (Oryza satiza L.) The main storage material in seeds, starch content and physicochemical properties, affect the quality of rice, and the accumulation level of rice starch also affects the yield of rice. Therefore, it is of great theoretical significance and practical value to clarify the key genes and regulatory networks in starch synthesis pathway of rice, a major food crop. In this study, the phenotype of Dianjingyou 1 grain silt mutant flo12 induced by MNU mutation was analyzed, and the target gene FLO12. was obtained by map-cloning. The main results were as follows: the appearance of endosperm was opacity after 1.flo12 seed was removed, and the cross-sectional observation showed that the endosperm was mainly silky inside endosperm. Scanning electron microscope (SEM) showed that the starch granules of wild type Dianjingyou 1 were polyhedron, compact and uniform in size, while the starch granules of mutant flo12 were regular and round, loose and varied in size, and there was a large gap between them. Compared with the wild type, the 1000-grain weight and grain width of flo12 decreased significantly. The semi-thin sections of endosperm showed that the powder structure of mutant flo12 was larger than that of wild type, and there were many small, scattered single starch granules. The results of physiological and biochemical indexes showed that compared with wild type, total starch content of flo12 decreased, amylose content decreased significantly, lipid content increased significantly, and urea dissolution characteristics of starch in flo12 mutant changed compared with wild type. 4. Obtaining FLO12. by Map cloning A hybrid combination of flo12 mutant and indica rice variety IR36 was prepared. The mutant gene was located in 93 kb region between LJ4-28 and LJ4-33 on chromosome 4 by F2 population. Gene sequencing showed that the 890 base pairs of Os04g0553000 in this region were mutated from C to T, leading to the mutation of 297 amino acids from Thr (threonine) to Ile (isoleucine). The complementary vector was constructed and transformed into flo12 mutants. The seeds of powdery endosperm with mutant phenotype and transparent endosperm seeds with restoring phenotype were harvested at the same time, and the ratio of transparent seeds to powdery seeds was in accordance with the 3:1 segregation ratio. Therefore, the gene Os04g0553000 is the gene that we are looking for to control the pollen endosperm phenotype of mutants. 6. 6. By analyzing the temporal and spatial expression of FLO12, it was found that the expression of FL012 was the highest in leaves because of the constitutive expression pattern, and the expression of FLO12 was also changed during endosperm development, and the highest expression was at 12 days after anthesis. It was predicted that the N-terminal of FLO12 protein contained a plastid-localized signal peptide. To verify this prediction, subcellular localization was performed in rice protoplasts. The results showed that the fluorescence signal of FLO12-GFP could co-locate with chloroplast autofluorescence, indicating that FLO12 was located in plastid. It was found that the expression of these proteins in wild type and mutant had no significant difference, which indicated that the mutation of FLO12 did not affect the expression of these proteins.
【学位授予单位】：南京农业大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：S511

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