以基因轉殖策略探討水稻OsATG8c與OsWRKY45之生理功能

Abstract

低溫往往影響水稻之產量與品質，是以低溫耐受性分子機制，尤其是低溫反應相關基因之功能探討益形重要。先前本實驗室已利用差異性表現法分離出28個受低溫誘導或抑制表現的基因（陳等，2000年）。本論文針對其中兩個基因OsATG8c（Osic1）及OsWRKY45（Osjc1）探討其基因表現。再以農桿菌轉殖方式分別在TNG67水稻過度表現或利用 RNAi 剔除此二基因，觀察轉殖株在不同逆境，如低溫或營養缺乏下之植株生理反應與農藝性狀；並了解此二基因在水稻生長發育過程所扮演之角色。 目前已知ATGs（autophagy）基因與細胞自噬過程有關，參予與在細胞內Cvt路徑（cytoplasm to vacuole targeting pathway）與自噬體路徑。在阿拉伯芥研究顯示自噬體相關基因與植物生長發育、生物逆境誘導PCD、營養逆境下提早老化及氮素缺乏下之生長過程至為相關。WRKY基因為植物所特有之轉錄因子超家族，可根據其WRKY domain與zinc-finger motif分為三群，與植物諸多生物/非生物逆境之反應、絨毛與種子的發育、糖的訊息傳遞有關。其中與本研究相關之group III群，在阿拉伯芥的研究顯示其多參與在病源菌防禦上游調控過程。 研究結果發現在水稻白化幼苗與低溫處理之三葉齡幼苗，雖此二基因均會受冷逆境誘導，但OsATG8c具有根、葉組織表現之差異性，而OsWRKY45則無。另外在不同生長發育時期，OsATG8c基因主要表現在抽穗前之花序，而OsWRKY45則會在老化葉片表現量增加。目前認為OsATG8c可能參與在水稻根部的ROS清除機制；而OsWRKY45則可能參與在生物與非生物逆境的上游訊息調控過程。為確切瞭解此二基因之功能，於基因轉殖部分已成功取得ABRC（ABA inducible）與RD29A（drought inducible）驅動OsATG8c表達之水稻轉殖株15株。此外以玉米ubiquitin起動子針對OsATG8c保守序列與3’UTR進行RNAi之轉殖株有16株。而Ubi:: OsWKRY45之3’UTR進行RNAi轉殖株五株，RD29A::OsWRKY45僅得一株。此些轉殖株經南方墨漬分析，大多具有1-2插入套數。另外取RD29A::OsATG8c之T1代殖株進行氮素缺乏逆境測試，初步並未觀察到明顯性狀差異；同時經RT-PCR證實ABRC::OsATG8c之T0代轉殖株在不需ABA前處理即可大量表現。未來研究擬針對T2代同質結合基因型OsATG8c與OsWRKY45的over expression及knockout轉殖株進行不同逆境處理，比較轉殖株與TNG67對不同逆境如低温、缺氮、病蟲害的反應與抗性，並觀察轉殖株於不同生長發育時期之變化。

The low temperature stress can greatly affect the quality and quantity of rice production, therefore to understand the molecular mechanism of low temperature tolerance in rice, especially the function of cold-associated responsive genes becomes very important. Previously, our lab had substrated out 28 genes either induced or repressed by cold from TNG 67 rice by differential display technique. In this paper, we first focus on two of the 28 genes, OsATG8c (Osic1) and OsWRKY45 (Osjc1) to study their genes expression pattern. Then with the Agrobacterium mediated transformation method, the gain-of-function (overexpression) and knock-out (RNAi) TNG 67 rice mutants of the above two genes were produced. The phenotypes and agronomic traits of these transgenic rice plants under different developmental stages or stress conditions such as nutrient limitation and low temperature stress will be determined. Through these studies, we aim to reveal how these two genes work and what are their physiological roles in different development stages and diverse stresses of rice. Currently, autophagy related genes (ATGs) are believed to be involved in the self-digestion processes of cells, that consisted of cytoplasm to vacuole targeting pathway (Cvt pathway) and autophagosome pathway. In Arabidopsis and Tobacco, the function of ATGs are demonstrated to participate in normal plant development, stress-induced differentiation, hyper-sensitive response induced programmed cell death (HR-PCD), accelerated senescence and shorter root formation under nutrient starvation. The WRKY superfamily are plant specific transcriptional factors, which are categorized in three distinct groups depending on the number of the WRKY domains and the pattern of the zinc-finger motifs. In Arabidopsis, the WRKY genes can response to most different biotic/abiotic stresses, trichome and seed development, sugar signal transduction. In addition, nearly all group-III members of Arabidopsis WRKY genes expression that related to our study are induced by diverse pathogens infection, suggests these WRKYs could be involved in the upstream regulation of plant-pathogen defense mechanism. The result showed that both the OsATG8c and OsWRKY45 genes expression can be induced by chilling in etiolated and three-leaf rice seedlings. However, OsATG8c displays differential gene expression in root and leaf tissues but not OsWRKY45. Besides, the OsATG8c transcript has the highest level in just heading inflorescence; the OsWRKY45 expression is enriched in the senescence process of leaf. Recent studies indicate that OsATG8c could be a player in the ROS removing mechanism of rice root; and OsWRKY45 may locate on the upstream of biotic / abiotic signaling pathway. In order to thoroughly understand the functions of these two genes, transgenic rices approach has been conducted. So far, fifteen ABRC::OsATG8c and RD29A::OsATG8c transgenic rice plants were obtained. In addiction sixteen rice plants regenerated from transformation with Ubi::OsATG8c（ORF and 3’UTR）. Five transgenic rice plants which carry the Ubi::OsWRK45 RNAi（3’UTR）construct were present, but only one line in RD29A::OsWRKY45. Southern blot analysis showed that most transgenic lines contain 1-2 copy of inserts. By the way, the T1 progeny of RD29A::OsATG8c had no observed growth phenotype under nitrogen-free culture solution. In T0 transgenic rice plants of ABRC::OsATG8c, the active expression of T-DNA introduced OsATG8c was detected by RT-PCR without pre-treated with ABA. In the future, the T2 homozygous progenies of overexpression and knock-out transgenic rice mutants will be continuous analyzed, including to measure the physiological responses, phenotypes changed as well as level of tolerance to different stresses such as low temperature, nitrogen starvation, pathogen infection etc.