糖及氮素對水稻基因表現的調控作用

Sugar and Nitrogen Regulation of Gene Expression in Rice

侯新龍(Shin-Lon Ho)

指導教授 ： 童武夫博士 ； 余淑美博士

國立臺灣師範大學/理學院/生物學系/博士(1999年)

摘要

無資料

關鍵字

水稻 ； 轉錄階層 ； 轉錄後階層 ； 訊息傳遞 ； 半胱胺酸內蛋白水解酵素 ； 啟動子 ； 農桿菌 ； 基因轉殖

並列摘要

英文摘要
In plants, carbohydrate and nitrogen metabolism are tightly-linked physiological functions and are essential for growth and development. The aim of the present study is to elucidate the mechanisms of sugar and nitrogen regulation of gene expression, which may help us better understand how plants respond to developmental and environmental cues during their life cycle.
First, to study the mechanisms that control sucrose-dependent gene expression, we performed differential screening of a cDNA library constructed from poly(A)+ mRNA prepared from 4 h sucrose-starved rice cells, and obtained six genes whose expression was sugar down-regulated (SD genes). Meanwhile, Northern blot analysis demonstrated that expression of several cDNAs, which encode proteins known to be required for cell growth, were sugar up-regulated (SU genes). Nuclear run-on transcription analysis and an assay with inhibition of mRNA transcription with actinomycin D demonstrated that expression of SU and SD genes in cultured rice suspension cells were differentially and coordinatelly regulated by sucrose at both transcriptional and posttranscriptional levels.
Next, to study the sugar signal transduction pathway, rice suspension cells were treated with sucrose, glucose, or glucose analogs. The results showed that glucose by itself or glucose and fructose hydrolyzed from sucrose all activated the expression of SU genes and suppressed the expression of SD genes. In constrast, the glucose analogs, 3-OMG (3-O-methylglucose) and 6-dG (6-deoxyglucose), which cannot be phosphorylated and metabolized by cells, did not activate the expression of all the SU genes but suppressed the expression of certain SD genes in sucrose-starved sells. Pyruvate suppressed the expression of certain SD genes, which suggests that the sugar signal transduction pathway overlap the metabolic pathway for gene repression. Study with protein kinase and protein phosphatase inhibitors, staurosporine and okadaic acid, respectively, demonstrated that protein phosphorylation and dephosphorylation were involved in sugar-regulated expression of SU and SD genes. In-gel kinase activity assays revealed that a 38-KD protein kinase activity might be involved in the suppression of the SD genes by sugars, while a group of 55-, 66- and 68-KD protein kinase activity might be involved in the activation of the SD genes under sugar starvation.
During the germination of cereal grains, sugar, amino acids, and small peptides derived from hydrolysis of endosperm nutrients are taken up by the embryos to support the growth of seedling. Cysteine proteinases (CysP) play a major role in the degradation of endosperm storage proteins. To investigate the significance of CysP expression in rice physiology and the regulatory mechanism of CysP expression, we isolated the genomic clones OsEP3A (Oryza sativa endoproteinase 3A). Genomic Southern blot analysis revealed that OsEP3A was a single copy gene and there were at least 5 to 7 genes encoding OsEP3A homologs in the rice genome. Northern blot anaylsis revealed that OsEP3A gene expression was preferentially expressed in germinating seeds and senescing leaves of rice. In cultured rice suspension cells, the expression of OsEP3A was up-regulated by nitrogen starvation. The OsEP3A promoter was linked to the coding sequence of a reporter gene, gusA, encoding b-glucuronidase (GUS), and stably introduced into the rice genome through an Agrobacterium transformation-mediated transformation system. The OsEP3A promoter was sufficient to confer the nitrogen metabolic regulation of GUS expression in cultured suspension cells. Histochemical study also indicated that the OsEP3A promoter was sufficient to confer the temporal, spatial and hormonal regulation of GUS expression in germinating seeds.
We also studied the expression pattern of a-amylase and CysP in the germinating seeds of a dicot plants, water spinach (Ipomoea auuatica Forsk). Accumulation of a-amylase and CysP mRNAs was aboundant in mature dry seeds. After the onset of germination, levels of both mRNAs in endosperm were enhanced and reached at peaks at day 0.5, then decreased rapidly to a very low level at day 3.5. However, no a-amylase mRNA was detected in axises and cotyledons of germinating seeds. The expression patterns of CysP mRNA were similar in both axises and endosperms during seeds germination. The accumulation of CysP mRNA in cotyledons increased gradually 0.5 day after imbibition, reached a peak at day 2.5, and then decreased sharply.
In plants, carbon and nitrogen are the two most important elements in maintaining cell structure and life. These two elements control expression of a variety of genes related to growth and metabolism. As cells are capable of maintaining a balance in ratio of carbon to nitrogen, suggesting that the synthesis and utilization of these two elements are tightly controled. It would be interesting to study in future whether there exist general mechanisms for sugar and nitrogen regulation of gene expression, and whether the sugar and nitrogen signal transduction pathways cross-talk in respond to changes in physiological and environmental conditions.

並列關鍵字

rice ； transcriptional level ； posttranscriptional ； signal transduction ； cysteine endoproteinase ； promoter ； Agrobacterium ； gene transformation

參考文獻

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