【摘要】：淀粉是水稻(Oryza satiza L.)種子中主要的貯藏物質(zhì),淀粉的含量和理化性質(zhì)影響稻米的各項(xiàng)品質(zhì)指標(biāo),同時(shí)水稻淀粉的積累水平還影響稻米產(chǎn)量。因此闡明水稻這一主要糧食作物的淀粉合成途徑中的關(guān)鍵基因和調(diào)控網(wǎng)絡(luò)具有重要理論意義與應(yīng)用價(jià)值。本研究以采用MNU誘變獲得的滇粳優(yōu)1號籽粒粉質(zhì)突變體flo12為研究材料,對其表型進(jìn)行分析,進(jìn)一步通過圖位克隆的方法獲得目標(biāo)基因FLO12。主要結(jié)果如下:1.flo12種子去殼后,胚乳外觀呈現(xiàn)粉質(zhì)不透明,橫截面觀察顯示主要是胚乳內(nèi)部粉質(zhì)。掃描電鏡觀察顯示,野生型滇粳優(yōu)1號的淀粉顆粒呈多面體,排列緊密,大小均一;突變體flo12淀粉顆粒呈規(guī)則圓形,排列松散,大小不一,顆粒之間存在較大間隙。與野生型相比,flo12的千粒重和粒寬極顯著下降。2.發(fā)育中期的胚乳半薄切片觀察顯示,突變體flo12造粉體結(jié)構(gòu)明顯比野生型大,并且存在許多小的、零散分布的單粒淀粉顆粒。3.生理生化指標(biāo)測定結(jié)果表明,與野生型相比,flo12總淀粉含量降低,直鏈淀粉含量顯著下降,脂質(zhì)含量極顯著升高,同時(shí)flo12突變體中淀粉的尿素溶解特性與野生型相比也發(fā)生改變。4.利用圖位克隆的方法獲得FLO12。配制了flo12突變體與秈稻品種IR36的雜交組合,利用F2群體將突變基因定位在第四染色體長臂標(biāo)記LJ4-28和LJ4-33之間的93 kb區(qū)域內(nèi)�；驕y序表明,該區(qū)間內(nèi)基因Os04g0553000的第890個(gè)堿基,由C突變?yōu)門,導(dǎo)致其編碼的第297個(gè)氨基酸由Thr(蘇氨酸)突變?yōu)镮le(異亮氨酸)。5.構(gòu)建互補(bǔ)載體轉(zhuǎn)入flo12突變體,在T0代植株上同時(shí)收獲到具有突變表型的粉質(zhì)胚乳的種子和恢復(fù)表型的透明胚乳的種子,且透明種子與粉質(zhì)種子的比例符合3:1分離比。因此,基因Os04g0553000就是我們尋找的控制突變體粉質(zhì)胚乳表型的基因。6.對FLO12的時(shí)空表達(dá)進(jìn)行分析發(fā)現(xiàn),FL012基因?yàn)榻M成型表達(dá)模式,在葉片中表達(dá)量最高;其中在胚乳發(fā)育過程中,FLO12表達(dá)量也有所變化,在花后12天表達(dá)量最高。7.預(yù)測發(fā)現(xiàn)FLO12蛋白N端含有一個(gè)質(zhì)體定位的信號肽。為了驗(yàn)證這個(gè)預(yù)測結(jié)果,在水稻原生質(zhì)體中進(jìn)行亞細(xì)胞定位。結(jié)果表明,FLO12-GFP的熒光信號能夠與葉綠體自發(fā)熒光共定位,說明FLO12定位于質(zhì)體中。8.對淀粉合成相關(guān)蛋白的表達(dá)進(jìn)行分析發(fā)現(xiàn),這些蛋白在野生型與突變體中的表達(dá)均無明顯差異,說明FLO12的突變并不影響這些淀粉合成相關(guān)蛋白的表達(dá)。
[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.
【學(xué)位授予單位】：南京農(nóng)業(yè)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2016
【分類號】：S511

【參考文獻(xiàn)】

相關(guān)期刊論文 前10條

1 朱昌蘭;賀浩華;賀曉鵬;彭小松;傅軍如;歐陽林娟;;稻米支鏈淀粉的結(jié)構(gòu)及其遺傳調(diào)控研究進(jìn)展[J];中國糧油學(xué)報(bào);2010年12期

2 鄭義;陸維忠;馬鴻翔;;植物淀粉生物合成的研究進(jìn)展[J];江蘇農(nóng)業(yè)科學(xué);2009年06期

3 陳峰;孫公臣;楊澤峰;張士永;張洪瑞;楊亞春;袁守江;張正球;;水稻淀粉合成相關(guān)基因?qū)Φ久灼焚|(zhì)效應(yīng)的研究[J];華北農(nóng)學(xué)報(bào);2009年02期

4 周琳;劉曉萌;陳龍;;植物淀粉顆粒結(jié)構(gòu)研究進(jìn)展[J];種子;2009年01期

5 何秀英;廖耀平;程永盛;陳釗明;陳粵漢;;水稻品質(zhì)研究進(jìn)展與展望[J];廣東農(nóng)業(yè)科學(xué);2009年01期

6 孫金才;楊澤敏;;灌漿期淀粉合成酶動(dòng)態(tài)與米質(zhì)的相關(guān)性分析[J];中國農(nóng)學(xué)通報(bào);2008年07期

7 康國章;肖向紅;王永華;郭天財(cái);朱云集;官春云;;小麥淀粉GBSSII基因的克隆、反義和RNAi載體的構(gòu)建[J];華北農(nóng)學(xué)報(bào);2008年01期

8 李春;宋廣芝;田紀(jì)春;;糯小麥及其Waxy基因的研究進(jìn)展[J];中國農(nóng)學(xué)通報(bào);2007年07期

9 高振宇,黃大年,錢前;植物支鏈淀粉生物合成研究進(jìn)展[J];植物生理與分子生物學(xué)學(xué)報(bào);2004年05期

10 徐富賢,鄭家奎,朱永川,王貴雄,楊大金,劉康;川東南雜交中稻超稀栽培對稻米整精米率和堊白粒率的影響[J];植物生態(tài)學(xué)報(bào);2004年05期

，

本文編號：2271975