Positional Cloning, Gene Expression, and Molecular Evolution of the Gene Conferring Purple Leaf Sheath in Rice

花青素可累積於水稻許多組織中，形成紫紅色或紫色外表性狀，以抵抗生物逆境和非生物逆境。亦不管是野生稻或是栽培稻皆可發現含花青素之紫色葉鞘，或不含花青素之綠色葉鞘其中稉稻台農72號之葉鞘為紫紅色，而秈稻台中秈17號之葉鞘為綠色，因此想瞭解影響水稻葉鞘顏色的基因為何。本研究以台農72號和台中秈17號之F2子代，利用定位選殖策略，找出在此分離族群調控水稻葉鞘顏色基因為OsC1，另在3個綠色葉鞘品種在OsC1調控區域R2R3 Myb序列有非同質性的SNP或序列產生缺失，序列差異造成水稻葉鞘顏色的改變。因而利用36個不同栽培品種、地方種與野生種探討OsC1基因之分子演化過程，藉由序列分析可歸類14種不同的單體型，並以中性理論Tajama’s D、 Fu and Li’s D 和 Fu and Li’s F檢測此基因，顯示OsC1可能受到天擇或選拔的影響。 為了證實OsC1基因影響著水稻葉鞘花青素之累積，挑選葉鞘顏色深淺不一的台農67號、台農72號、上季早稻和崑山五香稉等四個品種，測量花青素含量並以Real time PCR偵測OsC1和花青素相關基因OsB2、OsDfr和OsAns之表現量，結果呈現僅只有OsC1基因表現量與花青素含量有正相關，OsC1在深紫色葉鞘品種中葉鞘有較高的表現，並且隨著生育日數的增加營養生長時期基因之表現量也有顯著的提升，其他三個基因之表現與花青素含量則無相關性。由TNG72啟動子研究探討OsC1基因之表現位置，發現在三葉齡時期葉鞘無基因之表現，直到營養生長時期才有表現，因此可知OsC1確實影響著水稻葉鞘顏色之於不同生長時期變化。Anthocyanin can accumulate in several tissues in rice, leading to purple red or purple phenotypes for resistance to biotic and abiotic stresses. Purple and green leaf sheath with and without anthocyanin accumulation, respectively, can be found not only wild rice but also in cultivated rice. Japonica TNG 72 has purple leaf sheath, while indica TCS 17 has green leaf sheath. Based on the positional cloning approach, we used the F2 progenies of TNG 72 × TCS 17 to isolate the gene conferring purple leaf sheath in this segregating population, which was OsC1. Sequencing the OsC1 of 3 cultivars with green leaf sheath was found nonsynonymous SNP and deletion in the regulation region R2R3 Myb domain.. Thus, variation in OsC1 sequence changes the colors of the rice leaf sheath. In addition,, we used 36 accesions include cultivars, landraces and wild rice to study molecular evolution of OsC1, and neutrality tests, such as Tajama’s D, Fu and Li’s D, and Fu and Li’s F, revealed OsC1 alleles might be impacted by natural selection or artificial selection during rice evolution or domestication. To determine the correlation between OsC1 and the anthocyanin accumulation of leaf sheath, we used four culativars, TNG 67, TNG 72, Shang-Chi-Tsao-Tao and Kun-Shan-Wu-Siang-Keng with different levels of anthocyanin content to detect gene expression of OsC1, OsB2, OsDfr, and OsAns by real-time PCR. The results showed that gene expression of OsC1 only but not the other threewas positively correlated with anthocyanin content. Gene expression of OsC1 was high in varieties with purple leaf sheath and increased along development stages. By the GUS promoter assay, OsC1 did not express in the leaf sheath during the 3-leaf stage but did express until the vegetative stage. Thus, gene function and expression of OsC1 really affect the colors of rice leaf sheath in different development stages