水稻ABC類型ATP-Binding Cassette(ABC)轉運蛋白OsABCB10的功能分析

Functional Analysis of Rice MDR-type ATP-Binding Cassette (ABC) Transporter OsABCB10

林依潔(Yi-Chieh Lin)

指導教授 ： 洪傳揚

國立臺灣大學/生物資源暨農學院/農業化學研究所/碩士(2009年)

https://doi.org/10.6342/NTU.2009.01844

摘要

ABC轉運蛋白(ATP-binding cassette transporter)廣泛存在各種物種之間，對各種不同基質的轉運扮演重要的角色。目前植物的ABC轉運蛋白功能以阿拉伯芥研究較多，水稻中ABC轉運蛋白功能性分析的相關研究非常少。因此本試驗擬針對水稻ABC轉運蛋白MDR次基因家族成員OsABCB10基因進行功能性分析。胺基酸比對結果顯示，OsABCB10為全分子轉運蛋白，具有ABC轉運蛋白的保守性功能區域Walker A、ABC signature以及Walker B。即時定量聚合酶連鎖反應(Q-RT- PCR)分析OsABCB10組織專一性基因表現，結果顯示孕穗期水稻的葉身、葉鞘及花序中都有相當高OsABCB10基因的表現，未成熟種子中表現量更強。以植物荷爾蒙處理兩週大水稻幼苗，結果顯示NAA、JA及SA都會誘導OsABCB10基因在地上部或根部表現。高鹽、ABA以及低溫(4℃)等非生物逆境都會誘導地上部OsABCB10的表現，而根部則受高鹽、乾旱及低溫誘導表現，其中低溫處理可強烈誘導根部OsABCB10的表現超過1000倍以上。分析POsABCB10-GUS轉殖水稻，結果發現GUS主要累積在葉片以及未成熟花序中，尤其集中於內穎、外穎、小穗軸及雌蕊部分。以低溫處理POsABCB10-GUS轉殖株2天，結果顯示GUS受誘導表現累積於根部。將POsABCB10-GUS種子浸潤後，結果顯示浸潤後2小時啟動子活性最高，隨浸潤時間增加而活性降低。分析OsABCB10:GFP融合蛋白於細胞中表現的位置，結果顯示OsABCB10是一個表現於細胞膜上的蛋白質。進一步分析基因剔除突變株abcb10同型合子，外表性狀與野生型無明顯差異，但是溫室或是田間試驗的結果顯示，abcb10稔實率較野生型大幅降低60%以上，同時abcb10花粉也有近40%表現出發育不良的情形。反之，OsABCB10基因大量表現的轉殖系，則與野生型無明顯差異。這些結果顯示，OsABCB10可能是維持水稻穀粒稔實率的重要基因。另外，試驗中亦觀察到abcb10種子有發芽遲緩及發芽率降低的現象，顯示OsABCB10可能也參與了種子發芽過程的調節。此試驗的結果說明了低溫誘導表現的OsABCB10可能是參與調節水稻穀粒充實率或種子發芽的ABC轉運蛋白。

關鍵字

功能性分析 ； ABC轉運蛋白

並列摘要

ATP binding cassette (ABC) transporters ubiquitously existed in all living organisms, and play important roles in transporting various substances across membrane systems. The functions of ABC transporters have been investigated extensively in Arabidopsis, however, very few studies are reported in rice. To this end, this study focuses on the functional characterization of the rice OsABCB10 ABC transporter. Amino acid alignment analysis showed that the OsABCB10 is a full molecule MDR-type ABC transporter containing typical Walker A、ABC signature and Walker B domains. Quantitative RT-PCR analysis indicated that the OsABCB10 was highly expressed in blade, sheath and inflorescence at booting stage, and strongly expressed in the immature seeds. Treatment of two-week-old rice seedlings with different plant hormones revealed that the OsABCB10 was induced by NAA, JA and SA both in shoot and root. Furthermore, the OsABCB10 was slightly induced by salt, ABA and cold (4℃) in shoot tissue, while it was slightly induced by salt and drought in root tissue. Surprisingly, cold treatment dramatically increased more than 1000 folds of OsABCB10 transcript accumulated in root tissue. Transgenic rice harboring POsABCB10-GUS construct showed that GUS protein slightly accumulated in blade and inflorescence, and strongly expressed in palea, lemma, rachilla and pistil. Upon cold treatment, GUS protein was induced in root tissue of POsABCB10-GUS plants as compared with control plants. Moreover, OsABCB10 promoter activity was enhanced after 2 hours imbibition, and then gradually declined with imbibition time. Subcellular localization assay showed that OsABCB10 is localized at plasma membrane. The OsABCB10 knock-out mutant abcb10 showed a striking seed fertility reduced phenotype either cultivated in the greenhouse or in the field. Meanwhile, up to 40% non-fertile pollen was observed in abcb10. In contrast, rice overexpressing OsABCB10 had no effect on seed fertility. These results demonstrated that OsABCB10 is required for seed development, and may be a crucial gene to maintain seed fertility in rice. In addition, seeds of abcb10 showed a germination delayed phenotype and possessed lower germination rate as compared with WT, indicating that OsABCB10 plays a role in modulating seed germination. Taken together, our results provide evidences to support that cold treatment highly induced MDR-type ABC transporter OsABCB10 may be involved in mediating seed fertility or seed germination of rice.

並列關鍵字

Functional analysis ； ABC transporter

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