禾本科植物種子發芽時,alpha-澱粉水解酵素基因表現受 gibberellin
調節的機制, 一直是研究植物荷爾蒙調控基因表現機制的最佳材料之一 .
本實驗室過去由水稻發芽種子所構築成的 cDNA library 中篩選出四個
cDNA, 經兩端定序後, 發現其中一個 cDNA (aAmy10) 可能為尚未被發表的
基因. 本論文即將此 cDNA 的核酸序列完全定序. aAmy10 cDNA 的長度為
1471 bp, 經與相似性較高的 RAmy1A 的核酸序列比較, 只差 3 bp 即為
ATG 的位置. 以電腦估計其所合成的蛋白質共有 426 個氨基酸, 分子量為
47.6 kDa, pI 值為 4.83, 與 RAmy1A 所合成的蛋白質有 96% 的相似性.
在種子發芽時, 胚中的 aAmy10 基因在第二天開始表現, 至第六天達最多
量. 在糊粉層細胞中, aAmy10 基因的表現情形與胚類似. 因此在種子發芽
時 aAmy10 的產物可能參與分解胚及胚乳中澱粉的工作. 另外利用 8 個水
稻alpha-澱粉水解酵素 3' 端未轉譯區域 (3'- untranslated region) 的
基因專一性區域 (gene-specific region) 為探針, 觀察懸浮培養細胞在
缺糖狀況下與水稻種子在發芽時,alpha-澱粉水解酵素基因的表現情形. 在
有糖的狀況下, 只有 RAmy3E 的基因有少量的表現, 而在缺糖狀況下, 所
有的alpha-澱粉水解酵素的 mRNA 均會累積,其中以 RAmy3D 的累積量最多
, RAmy3E 次之, 兩者約佔缺糖時所有alpha-澱粉水解酵素 mRNA 的 90%.
在乾燥的種子的胚中, 只有少量的 RAmy1A 與 RAmy3E 的 mRNA. 當種子發
芽至第二天時, 胚中 RAmy3D 的 mRNA 的量稍微增加, 但至第三天時又迅
速減少. 發芽至第五天時, 在胚中 RAmy1A, RAmy1B, RAmy1C, RAmy3B 與
RAmy3E 的 mRNA 的相對量較多. 而在糊粉層中, 除了 RAmy3D 外, 所有的
alpha-澱粉水解酵素都會大量增加, 其中以 RAmy1A, RAmy1C, RAmy3B 與
RAmy3E 的量最多, 約佔所有alpha-澱粉水解酵素 mRNA 量的 90%. 而根與
幼莖中幾乎無法偵測得alpha-澱粉水解酵素的 mRNA. 我們的結果顯示, 在
有糖的狀況下, 可能主要由 RAmy3E 進行澱粉分解的工作. 在缺糖的狀況
下 , 細胞中澱粉的分解可能主要靠 RAmy3D 與 RAmy3E. 在種子發芽的初
期, RAmy3D 可能扮演分解澱粉的重要角色; 而在種子發芽的中, 後期, 胚
乳中澱粉的分解主要是由糊粉層所分泌的 RAmy1A, RAmy1C, RAmy3B 與
RAmy3E進行.因此至少有兩種因子可調控水稻alpha-澱粉水解酵素基因的表
現, 一是荷爾蒙 Gibberellin, 另一是糖.不同的因子刺激不同的alpha-澱
粉水解酵素基因的表現, 可能是水稻細胞應付不同生長環境的一種生理需
要,本研究提供研究生理功能的重要基礎 .

The regulation of alpha-amylase gene expression by gibberellins
during germination of cereal grain has been used as a model
system for studying the mechanism of hormonal regulation of gene
expression in plants. Previously our lab have cloned and
partially sequenced four rice alpha-amylase cDNA clones, sequence
of one of the cDNA clone (aAmy10) has not yet been reported. The
length of aAmy10 is 1471 bp, however, it is not a full length
cDNA clone. By comparison with another rice alpha-amylase gene
RAmy1A, aAmy10 probably miss 6 nucleotides which encoding two
amino acids at the 5 ' end. aAmy10 encodes a polypeptide of 426
amino acids with predicted molecular weight of 47.6 kDa and pI
value of 4.83. The homology of amino acid sequence between aAmy10
and RAmy1A is approximately 96%. In embryo, the accumulation of
aAmy10 transcripts first appeared at day 2 after germination and
reached its maximal level at day 6. The expression pattern of
aAmy10 in aleurone layer was similar as that in embryo. It
suggests that the gene product of aAmy10 is probably involved in
starch degradation during rice seed germination. Expression of
the alpha-amylase gene family in cultured cells starved of sucrose
and during seed germination was studied using eight rice alpha
-amylase gene specific probes. In cells grown with sucrose, only
the transcript of RAmy3E was detectable. After cells had been
starved of sucrose, transcripts of all alpha-amylase genes became
detectable. The expression level was highest for RAmy3D and
second for RAmy3E, which account for almost 90% of total alpha
-amylase transcripts in the starved cells. In embryos of dry
seeds, only low levels of RAmy1A and RAmy3E transcripts were
detectable. Five days after seed germination, the transcripts of
RAmy1A, RAmy1B, RAmy1C, RAmy3B and RAmy3E also accumulated in
embryos. In aleurone layers, the transcripts of all alpha-amylase
genes except RAmy3D increased with time for the first 5 days.
Transcripts of RAmy1A, RAmy1C, RAmy3B and RAmy3E account for
almost 95% of total alpha-amylase transcripts in aleurone layers
during germination. No alpha-amylase transcript was detected in
young shoot and roots 5 days after germination. Our results
suggest that in cells grown with sucrose, enzymes of RAmy3E is
probably the major alpha-amylase for starch degradation. In cells
starved of sucrose, enzymes of RAmy3D and RAmy3E become the two
major alpha-amylases for starch degradation. The gene products of
RAmy1A, RAmy1C, RAmy3B and RAmy3E are the major enzymes for
starch degradation during later stages of seed germination. Our
studies clearly demonstrate that different members of the alpha
-amylase gene family are differentially regulated in different
tissues as well as under different growth condition, which
provides a new insight into the physiological functions of these
alpha-amylase isozymes in rice.
The regulation of alpha-amylase gene expression by gibberellins