臺灣博碩士論文加值系統

中 文 摘 要 本實驗室曾分離出的水稻醇溶蛋白基因 (RP3) ,其啟動
子(1046 bp)與GUS結構基因所構築之嵌合基因 (chimeric gene) 已被送
入菸草中,並證實其表現具有種子專一性。一般影響基因表現與調控之因
子,包括(一)基因本身之結構、(二)生理/遺傳因子以及(三)環境因子。因
此關於水稻醇溶蛋白 (RP3) 基因表現與調控之研究,將從(1)縮減之RP3啟
動子,在水稻幼苗各部組織,進行短暫性基因表現試驗 (transient gene
expression);及(2)轉殖菸草懸浮細胞生理,生化特徵與GUS (-
glucuronidase)活性表現之關聯性兩方面加以探討,藉以找出和組織專一
性表現有關之啟動子區域,及與啟動子表現量有關之生理因子。 不同轉殖
菸草懸浮細胞品系 (lines 2、7及18),在繼代培養過程即表現GUS活性,其
量以line 2最高,line 7次之,而line 18則與pBI 101控制組相近。從外觀
形態觀察,line 2呈黃綠色,line 7淡黃色,line 18與pBI 101相似幾近乎
白色。 另一方面,不同轉殖菸草懸浮細胞品系,在繼代培養後過程,以
hexane: ethanol ( v/v=1/2 )抽取色素,經紫外光/可見光吸收光譜的掃
描,發現在663 nm和400 nm~500 nm間有顯著差異。其中具最高GUS活性的
line 2,在663 nm和430 nm有較大吸收值。line 7在663 nm和436 nm也有
較高吸收值;然line 18與控制組相似,而且在663 nm無吸收峰。從12天的
繼代培養過程中,我們發現line 2的GUS活性持續下降,而其色素在663 nm
和430 nm,扮隨著時間增加而減少。在line 7,其663 nm與 436 nm 隨著時
間增加而減少,但其 GUS 活性在繼代培養6天後開始增加。line 18與控制
組確無明顯變化。此結果顯示,在繼代培養過程中,line 2 GUS活性之變化
與色素量( 430 nm及663 nm )有關聯,而line 7及line 18則無明顯關聯。
此外,利用短暫性表現試驗及縮減試驗。將縮減後的啟動子,轉入水稻白化
幼苗的地上部,其結果發現啟動子長度為1046 bp (-1046 bp ~ -1bp) 與
縮減長度為664 bp (-664 bp ~ -1 bp) 部份不表現出GUS活性,然縮減長
度為437 bp (-437 bp ~ -1 bp) 與292 bp (-292 bp ~ -1 bp) 部份表現
出GUS活性。這似乎意味水稻醇溶蛋白基因 (RP3) 啟動子, 控制其組織專
一性之區域可能在-1046 bp到-664 bp的區段上。

ABSTRACT A rice prolamin gene RP3 was isolated from genomic
library of Oryza sativa cv. Tainung 67. The promoter region was
fused with bacterial -D-glucuronidase (GUS) for gene
expression and regulation study in transgenic systems. The GUS
activity was found exclusively in seeds of transgenic tobacco
regenerants. These data suggest the tissue-specific expression
of RP3 promoter in transgenic tobacco. Gene expression and
regulation are in general influenced by (1) gene structure (2)
physiological conditions/genetic background and (3)
environmental factors. Therefore, We focus on two aspects to
study rice RP3 gene expression and regulation. (1) Transient
gene expression of nest-deleted RP3 promoter/GUS chimeric gene
constructs in etiolated rice seedling. (2) Correlation of
biochemical/ morphological characteristies with RP3 promoter/GUS
chimeric gene expression in transgenic tobacco cell suspension
cultures. From transient gene expression, GUS activity was
only detected in shoots of etiolated rice seedling bombarded
with —437 and —292 chimeric gene constructs. The seed
specificity of RP3 gene expression in rice could be overcome.
These data suggest that the promoter area between —664 and
—437 may be required for regulating the specificity of RP3 gene
expression. Differential expression of GUS activity was
observed among transgenic tobacco cell lines. The highest
activity was found in line 2. Line 7 was in middle. And no
significant difference of GUS activity was detected between line
18 and negative control pBI101. The morphological variation was
also observed among transgenic cell lines. Line 2 is green-
yellow. Line 7 is light yellow, and white for line 18 and
pBI101. From spectrophotometric analysis, Two absorption peaks
around 663 nm and 430 nm were significantly increased in line 2.
The changes of the two peaks were positively correlated to the
changes of GUS activity in line 2 within a period of 12 days
after subculture. These data suggest that physiological
variations may exist and possibly affect directly or indirecty
the level of GUS activity in line 2.We conclude that cis-acting
elements of RP3 promoter regions and physiological conditions/
genetic background of transgenic tobacco may affect RP3 gene
expression and specificity. Identification and isolation of
factors such as specific motifs within RP3 promoter,
transcription factors and physiological components may help
address the issue of RP3 gene expression and regulation.