水稻粉質基因 FLO7 對穀粒成分及澱粉生合成相關基因之影響

The Effect of a Floury Gene Floury7 (FLO7) in Rice (Oryza sativa L.) on Grain Composition and Starch Synthesis Related Genes

林孟穎(Meng-Ying Lin)

指導教授 ： 林彥蓉

國立臺灣大學/生物資源暨農學院/農藝學研究所/碩士(2013年)

https://doi.org/10.6342/NTU.2013.10047

摘要

水稻穀粒呈不透明的突變體，包含粉質、糯性及半糯胚乳，為食品和工業利用上之重要性狀，不僅可作為研究充實期養分儲藏代謝過程的遺傳材料，也能促進了解澱粉生合成相關基因的調控網絡。本研究所使用的粉質胚乳材料為臺稉 8 號經 EMS 誘變且經多代自交固定而得之突變品系 WY14，其直鏈澱粉含量 (14.20 ± 0.05 %) 顯著低於臺稉 8 號 (18.15 ± 0.58 %)，第一期作穀粒充實較慢，最終累積乾重較輕，但在第二期作則無顯著差異。WY14 與臺稉 8 號之蛋白質種類及含量無顯著差異，而胚乳內脂肪酸含量和組成不同，WY14 總脂肪酸含量 (0.38 ± 0.03 %) 顯著高於臺稉 8 號 (0.09 ± 0.01 %)，棕櫚酸及花生酸比例顯著較高，且額外偵測到 0.16 % 長鏈飽和脂肪酸山萮酸。粉質突變基因經定位選殖的結果為 FLO7 的第一個外顯子有一核苷酸序列突變造成非同義置換，經基因及功能比對顯示 FLO7 為新穎的粉質基因，預測為 ABC 轉運蛋白，推測在水稻穀粒充實期澱粉生合成路徑中扮演運送物質的角色。以 RNA 干擾擾技術弱化 FLO7 之基因表現，22 個 RNAi 系之 T1 種子中，11 個轉殖系的榖粒透明與粉質分離，再度證明 FLO7 可造成水稻粉質胚乳。利用 3’ RACE 進行四種預測 FLO7 轉錄物之確認，FLO7 在胚乳內主要轉錄物為 FLO7-2a。以即時定量 PCR 反應進行 FLO7 及其他澱粉生合成相關基因於 101 年第一及第二期作水稻三葉齡組織或未成熟穎果的基因表現分析，FLO7 於三葉齡組織及未成熟穎果皆有表現，無明顯組織專一性，WY14 及臺稉 8 號於授粉後第 15 天表現量差異最大。以 WY14 相較臺稉 8 號之基因表現結果觀察，與澱粉生合成相關之上游路徑中，在細胞質作用的基因表現量大致呈現上升趨勢，如 UGP、OsAGPL2 及 OsAPGS2b 等；而下游路徑中，在澱粉體作用的基因表現量則下降，如OsGBSSI、OsSSIIIa 及 OsBEI 等。WY14 之 PPDKA 及 cyPPDKB 表現顯著高於臺稉 8 號，可能為脂肪酸含量較高之原因。推測 FLO7 可能參與在澱粉生合成之相關路徑中，影響特定物質轉運至澱粉體並導致澱粉排列改變。本研究結果對水稻胚乳內澱粉生合成相關基因調控機制及遺傳網絡的研究有所貢獻，日後亦可利用分子標幟輔助選種之方式將粉質胚乳性狀導入現行品種，增加稻米的多元應用。

關鍵字

水稻 ； 粉質胚乳 ； ABC轉運蛋白

並列摘要

Opaque rice endosperm including floury, glutinous, and dull grains is important for food and industrial applications. The corresponding mutants not only provide valuable genetic materials for elucidating metabolic processes related to nutrient storage during grain filling but also facilitate the understanding the gene network of starch synthesis related genes. A floury mutant, WY14, is one of the mutants from TK8 induced by EMS which possessed lower amylose content (14.20 ± 0.05%) compared to that of wild type TK8 (18.15 ± 0.58 %). WY14 proceeded worse grain filling and accumulated lower dry weight of grains in the first crop season of 2012 than TK8 did, but not in the second crop season of 2012. Profiles of total storage proteins in WY14 and TK8 revealed no difference. However, WY14 had higher amount of fatty acid and proportion of palmitic acid and arachidic acid than TK8, and WY14 possessed additional 0.16 % of long-chain saturated fatty acid, behenic acid. The mutated gene, conferring floury endosperm of WY14, was isolated by positional cloning, which was named FLO7 and annotated as ABC transporter.
The mutation of FLO7 responsible for floury endosperm was confirmed by RNAi. T1 seeds of RNAi lines showed segregation of both translucent and floury endosperm. Identification of 4 FLO7 transcripts by 3’ RACE suggested that the main FLO7 transcript in endosperm is FLO7-2a. FLO7 expressed not only in vegetative tissues but also in immature caryopsis, and FLO7 expressed differently with significance between WY14 and TK8 at 15 DAP. Gene expression of most starch biosynthesis related enzymes acting in cytosol had higher level, but not those in amyloplast which were down-regulated in WY14 as compared to in TK8.

Futhermore, higher expression of PPDKA and cyPPDKB may account for higher total amount of fatty acid in WY14 than in TK8. This research contributes to the relevant regulatory mechanism and genetic network related to starch synthesis in rice endosperm. The flo7 of WY14 may be applied to breed new floury varieties by marker assisted selection for multipurpose usage of rice grains.

並列關鍵字

Oryza sativa ； floury endosperm ； ABC transporter

參考文獻

李雅婷 (2012) 溫度逆境對不同穗位的乙烯合成及水稻穎果發育之影響。臺灣大學農藝學研究所學位論文

林鋐穎 (2009) 邁向水稻半糯基因 Du8 之定位選殖。臺灣大學農藝學研究所學位論文

曾盟群 (2012) 影響稻米味度及黏度特性之數量性狀基因座定位分析。臺灣大學農藝學研究所學位論文

廖英男, 劉景平, 林彥蓉, 吳永培 (2012) 水稻粉狀質基因 FLO6 之遺傳分析與初定位。嘉大農林學報 9: 1-19

劉瑋庭, 宋勳 (1996) 稻米品質的影響因素與分級。稻作生產改進策略研討會專刊臺灣省農業試驗所專刊: 133-153