水稻粉質基因 FLO7 之澱粉粒觀察、互補試驗及次細胞定位

Abstract

水稻是臺灣最重要的作物，以榖粒作為食用的部位，其中90% 以上都是澱粉質的胚乳，故澱粉粒的型態及排列是影響水稻的產量及品質重要的因子之一，提升米質則是水稻育種的首要目標，而不透明胚乳突變體的遺傳分析及定位選殖調控相關研究，可應用於提升米質及不同胚乳質地新水稻品種之育種。以EMS 誘變水稻臺稉8 號 (TK8)，從誘變庫篩選出一系列不透明的胚乳突變品系，依直鏈澱粉含量、澱粉粒型態可分為糯性 (waxy)、半糯 (dull) 及粉質 (floury)。利用掃描式電子顯微鏡觀察胚乳細胞，透明胚乳中充滿了緊密排列的多角型 (polygonal) 的澱粉粒構成複粒澱粉；乳白色糯性突變胚乳中多角形的澱粉粒內部，存在微小的孔洞及空隙；而不透明粉質胚乳則為排列疏鬆的圓形澱粉粒，澱粉粒之間有許多空隙。其中一粉質胚乳突變品系CNY921403 (WY14) 經定位選殖後發現非同義點突變發生在新穎的粉質基因FLO7 上，而FLO7 基因之啟動子及3’-UTR 區域的序列在秈、稉稻間有差異性。此基因註解為一ABC 運輸蛋白 (ATP-binding cassette transporter)，野生型 (wild type) 的FLO7 蛋白預測具有四個穿膜結構，突變品系因胺基酸改變發生在具高保守性的位點上，穿膜蛋白區域變為三個。為確認定位選殖結果，進行互補試驗將野生型FLO7 基因以基因轉殖方式導入突變品系WY14，T1 種子胚乳呈現透明及粉質的分離外表型，顯示FLO7 缺失確實造成粉質胚乳。轉殖融合螢光蛋白標誌之FLO7 蛋白於水稻原生質體，FLO7 蛋白表現於原質體、內質網及高爾基氏體。是以此新穎的粉質基因之功能性研究，提供更多關於影響胚乳發育及米質機制的研究線索。

Abstract Rice is the major crop in Taiwan, and the promotion of rice grain quality is an important goal in rice breeding programs. Because starchy endosperm is primary portion of rice grains, endosperm texture and starch granule morphology of rice grains are closely related to grain quality and yield. The comprehensive researches by a series of induced opaque endosperm mutants on genetic analysis, positional cloning and these isolated genes conferring to opaque grains provided new raw breeding material to improve rice grain quality and to breed new rice varieties for multipurpose usage of rice. The characteristics of the opaque endosperm mutants of TK 8 induced by EMS could be generally categorized into three types: waxy, dull, and floury grains according to the amylose contents and starch granule morphologies. The fracture surface of endosperm under scanning electron microscopy revealed that polygonal single starch granule was closely packed without space in translucent portions of endosperm, and the polygonal starch granules were tightly packed containing pores on the inside surface in the opaque portion of waxy mutants. In addotion, in the opaque sections of floury mutants, the single starch granules were round and loosely packed with a lot of air spaces. One novel opaque endosperm mutant line CNY921403 (WY14) exhibits a floury grain. By positional cloning, a nonsynonymous mutation occurred at FLO7 in WY14. The promoter and 3’-UTR region of FLO7 showed DNA diversity between japonica and indica varieties. FLO7 was annotated as a ATP-binding cassette transporter. Furthermore, the number of transmembrane domains of FLO7 protein was predicted 4 in wild type but only 3 in the mutated flo7, in which the nonsynonymous mutation resided in conserved domain among several species. To verify the positional cloning of FLO7, the complementary test was executed by transforming the wild type allele of FLO7 into mutant line WY14, and the T1 seeds of T0 plants were segregated for translucent and opaque endosperm, comfirming that FLO7 gene was responsible for flouy grain. By subcellular localization analysis by transforming the fluorescent protein-target FLO7 protein into rice protoplasts, FLO7 protein was observed in endoplasmic reticulum, Golgi apparatus and proplastids. As the result, the comprehsive research of this novel floury gene FLO7 provides additional information for the factors corresponding to endosperm development and rice quality.