Transgenic strategy for studying function of OsATG8c and OsWRKY45 genes in rice （Oryza sativa L.）
Date Issued
2005
Date
2005
Author(s)
Li, Chia-Yu
DOI
zh-TW
Abstract
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.
Subjects
自噬體
水稻
轉殖
autophagy
WRKY
rice
transformation
Type
thesis
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