Mechanism of Sugar Regulation of a-Amylase Gene Expression in Rice

• Main Authors: Lu Chung-An, 陸重安
• Other Authors: Su-May Yu
• Format: Others
• Language: zh-TW
• Published: 1998
• Online Access: http://ndltd.ncl.edu.tw/handle/24435546556171782211

博士 === 國防醫學院 === 生命科學研究所 === 86 === In germinating seeds and cultured suspension cells of rice, the expres sions of a-amylase genes are regulated by sugars and osmotica. To study the me chanisms of sugar and osmotica regulation at transcriptional level, I first tr ansferred the aAmy3 promoter- luciferase chimeric into rice using the Agrobact erium-mediated transformation. The results show that both sugars and osmotica inhibit the transcriptional activity of aAmy3 promoter. The regulatory elements in aAmy3 promoter was further analyzed by both loss- and gain-of-func tion studies using the protoplast transient expression system. The major sugar response sequence (SRS) was located between 186 and 82 base pairs upstream of the transcription start site. The SRS conferred sugar responsiveness to a min imal promoter in an orientation-independent manner. It also converted a sugar- insensitive rice actin gene (Act1) promoter into a sugar-sensitive promoter in a dose-dependent manner. Linker-scan mutation studies identified three essential motifs: the GC3 box (CCGACGCGG), G box (CTACGTG) and the TATCCA element, within the SRS. Sequences containing either the GC box plus G box or the TATCCA element each mediated sugar response, however, they acted synergistically to give a high level glucose starvation-induced expression. Using gel mobility shift assays, I demonstrated that nuclear proteins from rice suspension cells bind to the TATCCA element in a sequence-specific and sugar-dependent manner. SNF1 (sucrose-non-fermenting 1) plays an important role in sugar signal tranduction in yeast. It would be interesting to determine whether or not both the sugar respression of a-amylase gene expression in rice and the sugar respression of several genes in yeast act through similar SNF1-mediated pathway. A cDNA, RSNF1, encoding a putative homologue of the yeast (Saccharomyces cerevisiae) SNF1 has been isolated from a library prepared from rice suspension cell mRNA. Genomic Southern blot analysis revealed that the RSNF1 gene may exist as one or two copie in the rice genome. Northern blot analysis showed that it is constitutivel expressed in cultured suspension rice cells, germinating seeds, young shoot and root, and mature plant leaf and sheath. To determine the function of RSNF1, the RSNF1 gene was transferred into the yeast snf1 mutant and found to restore the SNF1 function. Finally , I stable transformed rice with the RSNF1 antisense DNA and preliminary result indicated that RSNF1 may be involved in the sugar signal transduction of a-amylase gene regulation.