利用水稻 T‐DNA 插入導致突變之基因選殖及功能分析‐穀粒發育異常突變株 48000A 之研究

Abstract

本研究主要在探討 T-DNA 插入突變株 48000A，具有種子稔實率偏低、榖粒重量較輕、萌芽勢較慢、株高較矮等外表性狀之可能原因。此突變株之 T-DNA 插在水稻第九對染色體中，在 BAC clone 中為 OJ1344_B01 BAC 上 147,606 bp 的位置，其插入點兩側 30 KB 間經 RiceGAAS 分析推測共有七個基因分佈: OA-25、 OA-27、OA-29、 OA-30、 OA-31、 OA-36、OA-40。本研究證實了其中有四個基因 OA-25、OA-29、OA-30、OA-31 均被活化，而此四個基因均為 putative flavonoid 3’-hydroxylase 基因 (putative cytochrome P450, F3’H)。因此本研究對此四個基因進行功能上的研究。OA-29、OA-31 主要可以在幼苗及成熟的葉片中偵測到，而 OA-25 及 OA-30 則否。再將此四個 F3’H 與其他已發表的 F3’H 進行胺基酸序列比對，可以發現僅有 OA-25 同時具有 p450 三個重要的保留序列: 膜鑲嵌位、鐵離子結合位以及酵素活性位，但是 F3’H 的保留序列卻與其他物種的不盡相同。已知 F3’H 之功能為將類黃酮素化合物碳環之 β-ring 進行氫氧基化，以產生更多的類黃酮素化合物，而類黃酮素的含量亦會影響植物株高以及授粉的功能。於是進一步的利用 ubiquitin 啟動子分別大量表現此四個 putative F3’H 基因於野生型 TNG67 植株並且對這些性狀做調查。結果發現轉殖株 Ubi:OA-29、Ubi:OA-30、Ubi:OA-31 種子發芽較晚，在幼苗時期只有 Ubi:OA-30 有類似於 48000A 的矮株以及短根的性狀; 而進入生長發育時期後，48000A 以及轉殖株 Ubi:OA-30 均有矮株高、低稔實以及花粉效力較低之性狀，初步認為基因 OA-30 有影響植物生長以及授粉的功能。再偵測其類黃酮素以及花青素的含量，48000A、Ubi:OA-29 均較 TNG67 為高，花青素的含量則以 48000A、Ubi:OA-29、Ubi:OA-30 較 TNG67 高，顯示基因 OA-29、OA-30 的確有影響類黃酮素、花青素的代謝途徑。過去的文獻中亦指出類黃酮素代謝路徑的基因可受光的調控，在光源較弱的環境下類黃酮素的累積量會減少。於是利用遮光處理觀察幼苗的生長狀況，可以觀察到 48000A 在遮光處理後的確有回復根長的現象，而四個基因 OA-25、OA-29、OA-30、OA-31 的活化程度也減弱了，推測此四個基因均有受到光的調控。

The purpose of this study is try to elucidate the possible cause of the delay germination, shorter plant height and lighter grain weight in a T-DNA insertion rice mutant 48000A. This mutant has its T-DNA inserted at the 147,606th base of BAC OJ1344_B01 on chromosome number 9 and seven annotated genes OA-25, OA-27, OA-29, OA-30, OA-31, OA-36, OA-40 were identified within a 30 kb region flanking to the T-DNA insertion site. Four of them OA-25, OA-29, OA-30, OA-31 were activated in this mutant and they were annotated as the putative Flavonoid 3''- hydroxylase (F3’H) genes. Flavonoid 3''- hydroxylase (F3’H) takes part in the hydroxylation pattern of the β-ring. Several flavonoid substrates such as naringenin, dihydrokaempferol, kaempferol and apigenin, can be hydroxylated by F3’H. Analysis of gene expression profile showed that OA-25 and OA-30 can hardly be detected while OA-29 and OA-31 can be detected in seedlings and leaves in TNG67. Amino acid sequence alignment revealed that only OA-25 contains all the three p450 conserved motives: membrane anchor site, Fe (II) iron binding site, and enzyme activity site, and all these four putative F3’H showed different in the two F3’H conserved motives. In order to study the function of these putative F3’H genes, transgenic lines Ubi:OA-29, Ubi:OA-30, Ubi:OA-31 driven by ubiquitin promoter were obtained. All transgenic lines showed delay germination, however, at the maturation stage, only Ubi:OA-30 revealed shorter plant height, low pollen viability and low fertility. Furthermore, 48000A and Ubi:OA-29 have higher flavonoid and anthocyanin contents. It is interesting to realize that the four activated F3’H genes in mutant 48000A were down-regulated under dark condition and showed longer root length.