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Copyright (c) 1990, Microsoft Corp 中 文 摘 要
從台農 67 號水稻白化苗中，分離出一種鹼性轉化一種結合性轉化兩
種酸性轉化 (IT I、IT II)。白化苗粗抽液經硫酸銨分劃、Con A 親和
層析 (不吸附部份)、DEAE-Sephacel 陰離子交換層析及製備式電泳可將
鹼性轉化純化至均質。鹼性轉化之最適作用 pH 值為 7.0 ，pI 值為
4.4液相膠體過濾層析測定其原態分子量為 240 kDa，以 SDS-膠體電泳測
定其次單元 60 kDa，所以鹼性轉化應為四元體結構。在基質特異性方
面，蔗糖為最佳基質 2.53 mM，能夠水解棉仔糖但對麥芽糖或乳糖則無作
用，因此為一種 b-fructofase。由於酵素不為 Con A 管柱所吸附，且無
法以醣蛋白染色法呈色，因此應不為醣蛋白。 其水解蔗糖產物 - 果糖為
競爭性抑制因子，而葡萄糖則為非競爭性因子。外加的蛋白質， 除了鹼
性磷酯 (alkaline phosphatase) 有活化效果外，其餘蛋白質 (如 BSA
，Con A 和尿素) 對活性皆無影響。Tris 對蔗糖轉化有很強的
抑制效果，一些金屬離子 (Pb2+、Zn2+、Cu2+、Hg2+ 和 Ag+) 和
能夠影響硫氫基的試劑對酵素的活性具有影響性， 因此鹼性轉化的活
性部位或其附近區域可能含有半胱胺酸殘基，直接或間接影響到對
蔗糖的水解作用。
抽取水稻白化苗 poly(A)+ RNA，利用 lZAP II 載體建立 cDNA 庫，由已
知的轉化 cDNA 推衍之胺基酸序列中，選取高保留的區域，合成
二段寡核酸作為引子 (primers)， 利用 RT-PCR 方法可合成一段 1.41
kb 的 DNA 片段，經定序證實為轉化 cDNA 的部分 序列，以此 DNA
片段作為探針，進行 cDNA 庫的篩選。篩選得到 17 株大小為
0.6 - 2.0 kb 的 cDNA，以 Southern 轉印分析證明其為轉化 cDNA。
依其對 Eco RV 、Pst I 與 Xho I 作用的相對位置，可分為 RIT1、
RIT2 與 RIT3 三類。經定序分析後發 現三者間轉譯區域的同質性高於
74%，非轉譯區域的同質性低於 43%。由於選殖到的 17 株 cDNA 皆
未包含轉化 cDNA 5''- 端序列，為了得到全長 cDNA，因此利用 5''
RACE 方 法進行 5''- 端 cDNA 的合成，然而僅得到 RIT1 5''- 端的序
列。RIT1 cDNA 全長為 2445 bp，ORF 為 1965 bp，可轉譯出含
有 654 個胺基酸的蛋白質。 為了探討 RIT1、
RIT2 與 RIT3 三種異構基因在水稻內的表現，以 PCR 的方法，合成對
RIT1 和 RIT2 具專一性的核酸探針，應用於基因表現的研究。當白化苗
在不同生長環境 刺激下，RIT1 和 RIT2 基因的表現各不相同：在厭氧
狀況下，RIT1 的表現量隨處理時間 的增長而下降，RIT2 的表現量於
12 小時便急速下降。RIT1 mRNA 量會隨光照時間增長而 持續增加，
RIT2 則在光照後 12 小時表現量達最大，隨後下降而持平。而在不同蔗
糖濃度 下，RIT1 的表現持續上升，但變化不大，RIT2 在 0.5%，4%
，8% 蔗糖濃度下分別出現表 現高峰，在 8% 蔗糖濃度下有最大的表現
量。 利用 T7 RNA
polymerase/T7 promoter 表現系統分別進行完整的 RIT1 cDNA，5'' 端與
3'' 端 RIT1 cDNA 在大腸桿菌中的表現。結果只有完整的 RIT1 cDNA 和
5'' 端 RIT1 cDNA 在 0.1 mM IPTG 的誘導下可得極少量具轉化
活性的蛋白質。此外，亦將完整的 RIT1 cDNA 進行在酵母菌中的表
現，於 30℃ 培養 60-70 小時可得較高之轉化活性， 表現量仍不高
。
Copyright (c) 1990, Microsoft Corp Abstract
One alkaline, one bound-form and two acid invertase activities
were detected in the shoots of etiolated rice (Oryza sativa)
seedlings. The alkalineinvertase (AIT) was purified to
homogeneity through steps of ammonium sulfatefractionation, Con
A-Sepharose affinity chromatography (non-retained), DEAE-
Sephacelchromatography and preparative electrophoresis. The pH
optimum of AIT was 7.0 and the molecular mass, determined by gel
filtration, was 240 kDa. It is apparently a homotetrameric
enzyme (subunit molecular mass 60kDa). The pI value was 4.4 as
determined by isoelectric focusing. The bestsubstrate of the
enzyme was sucrose, with a Km of 2.53 mM. It also
hydrolysesraffinose, but not maltose or lactose. So, it is a b-
fructofuranosidase. Theenzyme gave negative glycoprotein
staining. Of the hydrolysis products,fructose was a competitive
inhibitor while glucose was a noncompetitiveinhibitor. Treatment
with an alkaline phosphatase could activate AIT, whileother
proteins such as BSA, Con A and urease had no effect on the
enzymeactivity. The enzyme activity was inhibited by Tris and
metal ions (Pb2+, Zn2+,Cu2+, Hg2+, and Ag+). Thiol reagents
inhibit AIT activity.
1The cDNA library was constructed from mRNA purified from
etiolated riceseedlings by using lZAP II as vector. Two
oligonucleotide primers, corresponding to the two
conserved amino acid sequences of invertase found in other plant
species, were used for the reverse transcription polymerase
chain reaction. The resulting 1.41 kb cDNA was used as a probe
to screen the cDNA library. Seventeen positive clones were
isolated from 7 × 105 recombinant plaques. They were classified
into three types, RIT1, RIT2 and RIT3 , on the basis of their
relative Eco RV, Pst I and Xho I sites. The missing 5''-ends of
the RIT1 cDNA clones were obtained by the rapid amplification of
cDNA 5''-end (5''-RACE) technique. The complete nucleotide
sequence of RIT1 possessed open reading frame (ORF) of 1965 bp
encoding 654 amino acid residues. The coding regions of the
three cDNAs shared approximately 74% homology and the dudced
amino acid sequences were 80% homologous. Sequence comparison of
the three cDNAs revealed a homology below 43% in the 3''non-
coding regions. Two specific probes for RIT1 and RIT2 were
developed and used for analyzing the steady state levels of
mRNA at different organs and under some physiological stress
conditions. The expression level of RIT1 was decreased under
anaerobic treatment but increased by light treatment. Thelevel
of RIT2 mRNA was detected with little quanatity under anaerobic
treatment but increased quickly after 12 hours light treatment.
Both RIT1 and RIT2 genes were sucrose inducible. However, the
expression patterns were different. The results suggest that,
under some physiological stress conditions, the invertase
isogenes may be part of a system of cellular adjustment by
regulate sucrose storage, sucrose utilization and sugar
composition. To produce the invertase, and truncated
invertase in Escherichia coli under control of the T7 RNA
polymerase/promoter, the expression plasmid containing full
coding region, 5''-end or 3''-end of RIT1 cDNA were constructed.
Using pET-16b as vector and introduced into E. coli BL21(
DE3) for expression. However, expression of invertase
with enzyme activity was unsuccessful. The coding region
of RIT1 cDNA was also subcloned into plasmid pPICZaC, and
introduced into yeast Pichia pastoris for expression. Some
secreted protein with invertase activity was
obtained when cellls were grown and theexpression was induced
for 60-70 hours.
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